Bioavailability of a natural lead-contaminated invertebrate diet to zebrafish

Environmental Toxicology and Chemistry, Vol. 29, No. 3, pp. 708–714, 2010# 2009 SETAC

Printed in the USADOI: 10.1002/etc.61

BIOAVAILABILITY OF A NATURAL LEAD-CONTAMINATED INVERTEBRATE
DIET TO ZEBRAFISH
DAVID BOYLE,*y HEIDI AMLUND,z ANNE-KATRINE LUNDEBYE,z CHRISTER HOGSTRAND,y and NIC R. BURYyyKing’s College London, Nutritional Sciences Division, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, United Kingdom

zNational Institute of Nutrition and Seafood Research, P.O. Box 2029 Nordnes, Bergen 5817, Norway

(Submitted 6 August 2009; Returned for Revision 28 August 2009; Accepted 8 October 2009)

* T(david.

Pub(www.

Abstract—Dietary metals are increasingly recognized as key determinants of total metal burdens in fish, yet their ecotoxicologicalsignificance remains unclear. In this study, a pairwise experimental design was used to assess reproductive performance of zebrafish(Danio rerio) fed diets supplemented with a natural Pb-enriched polychaete, Nereis diversicolor. Zebrafish were fed 1% flake food (drywt diet/wet wt fish/d), 1% brine shrimp, and 1% N. diversicolor collected from either Gannel estuary, Cornwall, United Kingdom (UK),an estuary with legacy Pb contamination, or Blackwater estuary, Essex, UK, a reference site with low background metal concentrations,for 63 d. Mean daily dietary doses of Pb were 0.417 and 0.1 mg/kg/d (dry wt feed:wet wt fish) for fish fed N. diversicolor from Gannel andBlackwater estuaries, respectively. With the exception of Ag, which was higher for fish fed N. diversicolor from Gannel estuary, therewere no differences in daily dietary exposures to other metals (As, Cd, Cu, Fe, and Zn) between treatment groups. Fish fed Pb-enrichedGannel N. diversicolor exhibited no significant impairment to incidence of spawning, numbers of eggs per breeding pair or hatch rate ofembryos compared with pre-exposure levels, when N. diversicolor was omitted from the dietary regimen. Nevertheless, metal analysisrevealed significant increases in whole-body Pb burdens of male fish fed polychaetes from Gannel estuary, Ag in female fish fed Gannelworms, and Ag and Cd in male fish fed the Blackwater worms. These data demonstrate that Pb naturally incorporated in N. diversicolor isbioavailable to fish, and fish exhibit sex-dependent dietary metal accumulation patterns, but after 63 d of the experimental feedingregimen, reproductive performance was unaffected. Environ. Toxicol. Chem. 2010;29:708–714. # 2009 SETAC

Keywords—Aquatic invertebrates Metal bioavailability Fish Heavy metals Reproductive toxicity

INTRODUCTION

In recent years, there has been growing recognition that

deposit-feeding invertebrates assimilate and may thus mobilize

sediment-bound metals into aquatic food chains [1,2]. In estua-

ries, this pathway of metal transfer may be of particular sig-

nificance because estuarine sediments serve as sinks for

particulate metals of riverine origin: sorption of metals to

organic matter and subsequent deposition leads to net increases

in sediment metal concentrations over time [3]. Furthermore,

bioturbation by benthic invertebrates and vertical mixing of

sediment by tidal forces causes metals to be recycled into

surficial sediments and pore water for decades and longer after

a pollution event thus prolonging availability of metals to

estuarine fauna [4,5].

Sediment-bound metals have been shown to cause reduced

growth, survival, and reproductive output in benthic inverte-

brates [6,7]. Correlations between concentrations of metals in

estuarine sediment and tissue metal burdens in feral demersal

fish [8,9] and measurements of the assimilation of metal radio-

tracers from benthic invertebrates by fish in the laboratory [10]

suggest that the sediment-invertebrate-fish pathway of metal

transfer contributes significantly to total metal burdens in

estuarine fish. Establishing the ecotoxicological significance

of the trophic transfer of metals, a requisite for incorporating

dietary metals into environmental risk assessment, is however,

o whom correspondence may be [email protected]).lished online 9 November 2009 in Wiley InterScienceinterscience.wiley.com).

708

challenging for regulatory agencies [11]. Data collected from

metal-impacted feral fish populations will not discriminate

between the contributions of dietary and waterborne metal

exposure. Furthermore, laboratory studies that use artificial

diets to investigate metal toxicity in fish typically use metal

salts that are unreflective of metal species in prey items in

the field and may thus exhibit different bioavailabilities and

toxicities [12].

In our previous study [13], we used an estuarine polychaete,

Nereis diversicolor, collected from metal-impacted Restronguet

Creek as an experimental diet for fish. This approach presents

metals in ecologically relevant forms while minimizing simul-

taneous exposure to waterborne metals. Zebrafish fed these

polychaetes for 68 d exhibited reproductive impairments, most

markedly a 50% reduction in cumulative egg production,

associated with exposure to inorganic arsenic in tissues of

N. diversicolor. Moreover, growth of fish was unaffected, which

suggests that reproductive endpoints may be sensitive targets of

dietary metal toxicity.

The Gannel estuary, Cornwall, UK, has elevated Pb con-

centrations in sediments and estuarine fauna, including

N. diversicolor, as a result of discharges from abandoned

Pb mines in the watershed [14]. Waterborne Pb is a potent

reproductive toxicant to fish, affecting vitellogenesis and

spermatogenesis in salmonids at concentrations at and

below those permitted in the European Community dangerous

substances directive (20mg Pb/L) [15–17]. Lead in

N. diversicolor has been shown to be bioavailable to an

invertebrate predator [18]; however, its availability to fish

Bioavailability of a Pb diet to zebrafish Environ. Toxicol. Chem. 29, 2010 709

and its toxicological significance is not known and is the focus

of the present study.

MATERIALS AND METHODS

Fish husbandry

Adult zebrafish, of approximately 250 to 500 mg body mass,

were obtained from Neil Hardy Aquatica and acclimated to

deionized water supplemented with sea salts (60 mg/L Tropic

Marin1 termed synthetic water throughout the remainder of the

study) under a photoperiod regimen of 14:10 h light: dark and at

29� 18C. After two weeks’ acclimation, 15 male and 15 female

zebrafish were assigned to each of four aquarium tanks (dupli-

cate tanks per treatment) containing 40 L of continuously

aerated and filtered synthetic water and with conditions as

described above. At least 50% of the water was renewed every

2 d.

Collection of Nereis diversicolor

Nereis diversicolor were collected from intertidal mudflats

in an unimpacted area, Blackwater estuary, Essex (N 051 44 08,

E 000 41 34), and from the metal impacted Gannel estuary,

Cornwall (N 050 24 31, W 005 05 172), UK, and transported

back to the laboratory in sediment from source. Nereis diversi-color were individually depurated for 72 h in containers with

100 ml seawater (15%) at 158C to remove sediment from

the intestinal tract of the worms so that zebrafish were exposed

only to biologically incorporated metal. After depuration,

N. diversicolor were rinsed with deionized water to remove

external salt and stored at �808C until required.

Dietary exposure

For the duration of the acclimation period and for 14 d before

the onset of metal exposure (the pre-exposure period), zebrafish

were maintained on a diet of 1% Tetramin flake food (Tetra) and

2% brine shrimp (Artemia sp., from frozen, Ruto, The Nether-

lands; dry wt diet/wet wt fish calculated from initial total wt of

all fish in each tank). Baseline reproductive performance of fish

in each of the four tanks was assessed during this pre-exposure

period. Subsequently, zebrafish were switched to experimental

diets; equal dry weight rations of flake food, brine shrimp and

N. diversicolor (minus head because the jaws of N. diversicolorare unpalatable and contain high concentrations of metal

unlikely to be bioavailable to predators [19]) from Gannel

estuary or Blackwater estuary, at a combined rate of 3% dry

weight diet/wet weight zebrafish/d. Each dietary constituent

was fed to zebrafish in separate feeds: polychaetes (morning),

Table 1. Metal concentrations of dietary

Ag As

Nereis diversicolor (Gannel)ac 2.8� 0.9A 18.3� 0.3A 0.26�Nereis diversicolor (Blackwater)ac 0.71� 0.02B 15� 0.7B 0.46�Flake fooda 0.37� 0.05 141� 35 1.3Brine shrimpa 0.14� 0.02 8.1� 1.4 0.37Dose (Gannel)b 0.01–0.07 1–2.2 0.01Dose (Blackwater)b 0.005–0.01 0.95–2.2 0.01

a Data are given as means� standard error of three replicates (mg/kg dry wt).b Data are minimum and maximum daily dietary doses (mg/kg/day, dry wt diet/we

being fed 1% flake food, 1% brine shrimp, and 1% N. diversicolor per day.c Different uppercase letters denote significant difference between polychaetes at

flake food (afternoon), and brine shrimp (evening). Zebrafish

from both treatments were fed a diet containing flake

food and brine shrimp to adjust for possible imbalances in

nutritional value of N. diversicolor. Metal concentrations of

N. diversicolor, flake food, brine shrimp, and daily dose of

dietary metal during the exposure period are shown in Table 1.

Reproductive performance

A pairwise experimental design, comparing reproductive

performance within tanks before and after metal exposure,

was used to investigate the effects of N. diversicolor on

reproduction of zebrafish. After 7 and 14 (day 0 of the exposure

period) days of the pre-exposure period and on days 21, 35, 49,

56, and 63 of the exposure period and after the third feed (brine

shrimp), 9 or 10 pairs of zebrafish from each tank (18–19 pairs

per treatment) were each removed to separate 2-L marble

bottomed beakers filled with 1.5 L synthetic water. Before

feeding the following day, zebrafish were returned to their

respective tanks and embryos collected. The number of pairs

of spawning fish and the numbers of embryos produced were

recorded. A representative sample of 50 embryos per pair was

removed to Petri dishes with 50 ml of synthetic water and

0.0002% w/v methylene blue [20], an antifungal agent, and

incubated at 30� 18C until hatching. Assessments of reproduc-

tive performance were separated by at least 7 d.

Metal analysis

After the final spawn, zebrafish were not fed for 2 d to

evacuate the gastrointestinal tract, a transit time ascertained

as that required to remove all trace of the last meal (Boyle,

Hogstrand, and Bury, unpublished data). After an overdose

of benzocaine (150 mg/L; Sigma-Aldrich), zebrafish were

weighed, liver excised and weighed, and then the combined

carcass and liver were dried to constant weight at 808C.

Zebrafish and N. diversicolor (minus head) were digested with

6 N Ultrapur HNO3 (Merck) for 12 h and made up to volume

with Milli-Q1 ultrapure water. Water samples (n¼ 3 per tank)

were collected after the third feed of the day and before a

50% water change to ascertain maximum waterborne levels of

metals. Samples were analyzed for Ag, As, Cd, Cu, Pb, and Zn

using an inductively coupled plasma-mass spectrometer (ICP-

MS, PlasmaQuad PQ2 Turbo; Thermo Elemental) and Fe using

atomic absorption spectrometry (AAS, SpectrAA-50; Varian).

Stock standards (10 g/L) of the metallic elements were prepared

in 2% v/v nitric acid (Trace Analysis Grade; Fisher Scientific).

Standards were obtained from the following: As, from Aldrich;

Ag, Cd, Fe, Zn, In, and Ir, from BDH; and Cu, from Fisher

constituentsa and daily metal doseb

Cd Cu Fe Pb Zn

0.02B 48.6� 3.8 412� 123 33.4� 4.6A 127� 120.04A 39.1� 1.2 695� 55 1.7� 1.1B 109� 14

� 0.4 104� 19 1743� 416 1.2� 0.3 1237� 263� 0.07 18.8� 2.8 1066� 109 7.1� 0.9 123� 19–0.03 1.3–2.1 20–44.8 0.3–0.48 9.8–19.3–0.03 1.2–2 21.5–44.2 0.07–0.14 9.2–19.2

t wt fish) based on metal concentrations in dietary constituents and zebrafish

p< 0.05.

710 Environ. Toxicol. Chem. 29, 2010 D.Boyle et al.

Scientific. Indium and Ir were added as internal controls to

monitor instrumental drift during ICP-MS analysis. Triplicate

analysis was performed for each sample and a mean obtained.

In addition, certified reference materials were included for

the analysis of total metals in N. diversicolor. For example,

obtained values for Pb from oyster tissue (SRM 1566b; National

Institute of Standards and Technology) and lobster hepatopan-

creas (TORT-2; National Research Council Canada) were

0.29� 0.015 mg/kg, n¼ 4 and 0.43� 0.05 mg/kg, n¼ 4 and

agreed well with the certified values, 0.31� 0.01 mg/kg and

0.35� 0.13 mg/kg, respectively.

Arsenic speciation analysis in N. diversicolor

Total As and arsenic species was quantified in pooled freeze-

dried samples of N. diversicolor by ICP-MS and anion and

cation exchange high-performance liquid chromatography-ICP-

MS according to protocols previously described [21,22] and as

used by Boyle et al. [13].

Data handling and statistical analysis

Statistical analysis was performed using Graphpad prism,

version 5.01 for Windows1 (http://www.graphpad.com). Dif-

ferences in spawning frequency, normalized to pre-exposure

levels, was assessed between tanks and also treatments with

linear regression analysis. Statistical analysis was performed on

a tank basis; however, for ease of visualization, both tanks

within each treatment are combined to give single data points

for egg production per spawning pair and percentage hatch rate

at each time point. Egg production per spawning pair and

percentage embryo hatch rate (arcsine-transformed) were com-

pared within tanks to pre-exposure performance with one-way

analysis of variance followed by Dunnett’s test, or, where

transformation failed to correct non-normally distributed data,

with Kruskal-Wallis test followed by Dunn’s test. Metal bur-

dens in whole bodies of zebrafish, liver somatic index (LSI) and

zebrafish weights were compared between treatments using

Student’s t test, following log transformation of non-normally

distributed data where effective and with Mann-Whitney U test

otherwise (tanks within treatments were not significantly differ-

ent and were combined). A p value of <0.05 was considered

significant.

RESULTS

Metal content of polychaetes and experimental diets

Nereis diversicolor from Gannel estuary exhibit elevated

Ag, As, and Pb but significantly lower Cd compared with

N. diversicolor from Blackwater estuary (Table 1). Measured

Pb burdens in N. diversicolor from Gannel estuary and Black-

water estuary were 33.4� 4.6 and 1.7� 1.1 mg/g dry weight

(mean� standard error; n¼ 3), respectively. Lead was also

Table 2. Arsenic speciationa

Total As Inorganic Asb

Gannel estuary 18.3� 0.3A 0.8� 0.1A (3.9� 0.5%)Blackwater estuary 15� 0.5B 0.1� 0.05B (0.7� 0.3%)

a Data are means� standard error of three pooled samples (mg/kg dry wt). Values iAs. AB¼ arsenobetaine, TETRA¼ tetramethylarsonium ion. Different uppercas

b Inorganic As is the sum of As3þ and As5þ.

detected in both brine shrimp, 7.13� 0.94 mg/g, and flake food,

1.24� 0.29 mg/g (mean� standard error; n¼ 3), amounting to a

combined daily dietary dose of 0.3 to 0.48 and 0.07 to 0.14 mg

Pb/g zebrafish (wet wt), based on minimum and maximum

concentrations measured in constituent dietary components for

fish fed Gannel or Blackwater estuary polychaetes, respectively.

The daily dose of Pb and other metals fed to each group of fish is

shown in Table 1.

Arsenic speciation analysis of N. diversicolor

Nereis diversicolor collected from both the Gannel and

Blackwater estuaries retained a small percentage, 3.9 and

0.7%, respectively, of total As in inorganic forms (AsIII and/

or AsV) (Table 2). Organic As species detected included

arsenobetaine (15.1 and 35.7%) and tetramethylarsonium ion

(TETRA; 6.4 and 0.2%) in worms from Gannel and Blackwater

estuaries, respectively. Other arsenicals present in Blackwater

polychaetes include dimethylarsinate (DMA), arsenocholine

(AC), and trimethylarsoniopropionate (TMAP); however

levels, were close to the detection limits and concentrations

were not quantified. The Gannel N. diversicolor did not contain

AC, while DMA, dimethylarsinoylethanol (DMAE) and four

unknown arsenicals were detected but not quantified.

Water metal concentrations

Where detectable, unfiltered waterborne metal concentra-

tions did not differ significantly between treatments with the

exception of As, which was higher in tanks containing fish fed

N. diversicolor from Gannel estuary (2.29� 0.21 mg/L) com-

pared with Blackwater estuary (1.55� 0.24 mg/L). Lead was

not detected in the tank waters at any time during the exposure

(<1.46 mg Pb/L) and Pb burdens in fish are, therefore, expected

to originate from the diet.

Zebrafish mass and LSI

No significant difference was found between wet weight

(, Blackwater 0.55� 0.03 g, Gannel 0.56� 0.03 g, and <Blackwater 0.41� 0.02 g, Gannel 0.45� 0.02 g; mean� stan-

standard error, n¼ 11–13, p> 0.05) and LSI (, Blackwater

2.03� 0.29%, Gannel 2.23� 0.2%, and < Blackwater 0.63�0.1%, Gannel 1.01� 0.18%; mean� standard error, n¼ 11–14,

p> 0.05) of male or female fish following final spawn at day 63

and 2 d of depuration.

Reproductive output

Mean number of eggs per spawning pair and embryo hatch

rate were measured at time points during the 63-d exposure and

compared with reproductive performance during a pre-exposure

period (Fig. 1). There was no effect of estuary of origin of

N. diversicolor on percent hatch rate and mean hatch success

was high, exceeding 75% during both the pre-exposure and

in Nereis diversicolor

AB TETRA

2.8� 0.1B (15.1� 0.4 %) 1.2� 0.1A (6.4� 0.5%)5.4� 0.4A (35.7� 2.4%) 0.03B (0.2%)

n parentheses are measured concentrations expressed as a percentage of totale letters within columns denote significant difference at p< 0.05.

Fig. 1. Mean number of eggs (A) and % hatching success of embryos (B) per spawning pair of zebrafish fed Nereis diversicolor from Gannel estuary (open bars)and Blackwater estuary (closed bars). Data are means� standard error (n¼ 7–28), from two replicate tanks per treatment. Day 0 data are combined data from twotime points within a pre-exposure period.


exposure periods. There was no significant negative effect of

N. diversicolor from Gannel estuary on the mean number of

eggs produced per spawning pair. There was, however, con-

siderable variation in clutch sizes between females, which

ranged from 87 to 1,233 eggs, and between time points within

tanks. One tank from each treatment exhibited a significant

increase in mean number of eggs per spawning pair at one time

point (day 49 and 56, in one tank fed Blackwater and Gannel

worms, respectively) during the exposure but the effect was

transient and output returned to pre-exposure levels by day 63.

This variability between tanks was also evident in the spawning

frequency of fish, but there was no significant difference

between replicate tanks or treatments when data was normal-

Fig. 2. Spawning frequency (%) per tank of zebrafish fed Nereis diversicolorfrom Gannel estuary (open bars) and Blackwater estuary (closed bars)(n¼ 2). day 0 data are mean spawning frequencies during a pre-exposureperiod (n¼ 4 per treatment).

ized to pre-exposure levels and analyzed by multiple linear

regression analysis (Fig. 2).

Zebrafish tissue metal analysis

Metal concentrations were analyzed in carcasses of zebrafish

at the end of the 63 d dietary exposure. A significant increase in

Pb concentrations in whole bodies of male fish was observed as

well as a close to significant elevation in female Pb burdens

(p¼ 0.062, Student’s t test, n¼ 11–13) of zebrafish fed

N. diversicolor from Gannel estuary (Fig. 3). There was no

significant difference in tissue residue concentrations of As, Cu,

Zn, or Fe ( p> 0.05, Student’s t test, n¼ 11–13). Significant

differences were noted in Ag burdens in fish between the

treatments; however, the trends were not consistent between

the sexes. Ag burdens were higher in male fish fed Blackwater

worms than Gannel worms (0.009� 0.001 and 0.006�0.001 mg/g wet wt, respectively), but significantly lower in

female fish (0.005� 0.001 and 0.007� 0.001 mg/g wet wt,

respectively), despite Ag burdens being more than 10-fold

lower in Blackwater worms. Cadmium burdens in male fish

fed Blackwater polychaetes were also significantly higher.

Differences were also evident in metal burdens between sexes;

Fe, Pb, and Zn were higher in male fish, whereas As tissue

concentrations were higher in female fish from both treatments.

DISCUSSION

The objective of the present study was to determine if Pb

from a natural metal-contaminated invertebrate diet was bio-

available to fish and to examine its effect on reproduction.

Previous studies have investigated the effects of Pb exposure in

fish using artificial feeds [23,24] and diets formulated from

chironomids [25] or other fish [26,27] that have been exposed to

Pb in the laboratory. Using a different approach, the present

study is one of only a handful to use invertebrates collected from

the field as experimental diets, thereby presenting Pb in eco-

logically relevant forms [13,28].

Total Pb tissue burdens were significantly elevated in male

fish, and there was a trend (although insignificant) toward

Fig. 3. Whole-body metal burdens (mg/kg wet wt) of male (A) and female (B) zebrafish fed a natural metal contaminated diet for 63 d from Gannel estuary (openbars) or Blackwater estuary (closed bars). Data are presented as means� standard error (n¼ 11–14). Different upper case letters denote significant differencebetween treatment groups (Student’s t test or Mann-Whitney U test, p< 0.05).


increased Pb levels in female fish fed N. diversicolor from

Gannel estuary. With regard to other studies of dietary Pb

exposure in fish, the mean daily dose of Pb fed to zebrafish

in the present study was low, comparable to the lowest dose

used in the study of Alves et al. [24], which was observed to

cause transient disturbances to plasma ion balance in rainbow

trout. Moreover, because there was only a fourfold difference in

dietary Pb doses between treatment groups in the present study

and Pb burdens have been observed to stabilize or decrease in

fish fed Pb-enriched diets for long durations [23], significantly

elevated Pb concentrations in carcass of male fish fed for 63 d

may thus be indicative of a high trophic availability of Pb in

polychaetes from Gannel estuary. Metals in invertebrates col-

lected from the field are present as complex mixtures of species,

for example protein-bound metal, which may exhibit different

bioavailabilities compared with the free metal ion [29]. The

biological form of Pb in N. diversicolor from Gannel estuary is

yet to be determined; however, in conjunction with the study of

Rainbow et al. [18], who demonstrated the trophic transfer of Pb

from N. diversicolor to the invertebrate predator Nereis virens,

these results demonstrate that dietary exposure may be a source

of Pb for predators of N. diversicolor in Gannel estuary.

Zebrafish fed Pb-contaminated N. diversicolor from Gannel

estuary for 63 d exhibited no significant impairment to repro-

ductive performance. Measures of embryo hatch rate, egg

clutch size, and the frequency of spawning were unaffected

by dietary Pb exposure when compared with pre-exposure

levels; however, considerable variation, both within and be-

tween treatments, was evident at tank level. The inclusion of a

suitable pre-exposure period in which baseline performance

was assessed before dietary metal exposure was, therefore,

essential to the experimental design [30].

Few studies have investigated reproductive endpoints of

dietary Pb exposure in fish. Rademacher et al. [26,27] reported

altered concentrations of neurotransmitters, including dopamine

and serotonin, in brain regions of rainbow trout (Oncorhynchusmykiss) fed Pb-enriched fathead minnow (1.5 mg Pb/kg body wt

trout/d) for 14 d. Dopamine and serotonin modulate the activity

of gonadotropin releasing hormone in the hypophysis of fish and

perturbations to levels of these neurotransmitters may affect

functioning of other organs in the reproductive axis; release of

gonadotropin II, the maturation hormone in fish, from pituitaries

of Atlantic croaker (Micropogonias undulatus) was reduced in

fish fed 15 mg Pb/kg body weight/day (shrimp/trout chow diet

containing PbCl2) for 30 d [31].

Of interest, none of these studies report tissue concentrations

of Pb; however the doses used in the studies of Rademacher

et al. [26,27], 1.5 mg Pb/kg body weight trout/day, are approxi-

mately 3 to 5 times higher than the daily dietary dose given to

zebrafish in the present study. Because the brain is not a

principle tissue for Pb accumulation; 0.1% of total accumulated

Pb was measured in brains of rainbow trout fed enriched trout

pellets (500 mg Pb/kg) [23], this suggests that the brain may be

highly sensitive to Pb.

Arsenic speciation analysis revealed similar chemical pro-

files in polychaetes from both estuaries. Arsenic toxicity is

dependent on chemical form, with inorganic forms, As3þ

and As5þ, most often associated with highest toxicity, including

reproductive disturbances [13,32,33]. We have previously

reported low inorganic As tissue concentrations and a high

percentage composition of organoarsenicals in Blackwater

worms [13] and the absence of significant quantities of

inorganic arsenic in Gannel estuary polychaetes supports the

hypothesis that biotransformation to organoarsenicals is the

typical mechanism of inorganic As detoxification for

N. diversicolor during low level As exposure [34].

Silver concentrations were elevated in male fish fed Black-

water worms, despite significantly lower daily dietary exposure.

The reverse was, however, evident in female fish. Total metal

concentrations in prey items are often poor indicators of bio-

availability; Ag in Blackwater worms is found mainly in a

cytosolic protein fraction that has been associated with high

trophic availability [35]. Furthermore, fish exhibit gender-spe-

cific reproductive and physiological nutritive requirements. For

example, the egg-yolk precursor protein vitellogenin is a Zn-

containing protein [36]. This may affect the assimilation of

nonessential metals by means of increased uptake of protein-

bound metal or by means of shared sites of uptake with essential

metals [37]. Differences in the metal burdens of male and

female fish were apparent for most metals measured in the

present study and have been widely reported in wild fish

populations [9]. The toxicological significance of Ag exposure


in the present study is unclear; however, tissue burdens are low,

below those reported in laboratory maintained control fish used

in a dietary Ag study [38] and unlikely to impact health of fish in

the present study.

The lack of a discernible effect on reproduction suggests that

the dose of Pb from the diet (0.3–0.48 mg Pb/g zebrafish (wet

wt)/day) (Table 1) was within the detoxifying capacity of

zebrafish. The feeding regimen chosen meant that the nereid

worms formed only part of the diet (33.3%). In a recent study

using N. diversicolor from an estuary with elevated arsenic,

Boyle et al. [13] observed an effect on reproductive output of

fish fed 100% diet of polychaetes. Consequently, a further study

where the proportion of the Pb-contaminated worms in the diet

is increased would be required to ascertain if dietary Pb acts as a

reproductive toxicant and if this route of uptake may contribute

to Pb-associated reproductive disturbances observed in fish

populations in the field [39,40].

Acknowledgement—D. Boyle was supported by a PhD studentship from theFisheries Society of the British Isles. We thank Richard Handy (University ofPlymouth) for assistance with the metal analysis. The experiment wasperformed according to local animal care regulations.

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Bioavailability of a natural lead-contaminated invertebrate diet to zebrafish

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