Accumulation of Heavy Metals in Different Body Tissuesof Atlantic Salmon, Salmo salar L., Exposed to a Model Mixture(Cu, Zn, Ni, Cr, Pb, Cd) and Singly to Nickel, Chromium,and Lead

Gintaras Svecevicius • Gintar _e Sauliut _e •

Raimondas Leopoldas Idzelis • Joana Grigeleviciut _e

Received: 26 July 2013 / Accepted: 24 February 2014 / Published online: 1 March 2014

� Springer Science+Business Media New York 2014

Abstract One-year-old Atlantic salmon smolts were

exposed for 2 weeks either to six priority heavy metal

mixture or to Ni, Pb, and Cr singly at a concentration

corresponding to Lithuanian inland water standards: Cu

– 0.01, Zn – 0.1, Ni – 0.01, Cr – 0.01, Cd – 0.005 and

Pb – 0.005 mg/L, respectively under semi-static condi-

tions. The presence of metal mixture in the water only

partly (by 50 %) affected the accumulation of single

metals in body tissues (muscle, gills, liver and kidneys)

probably due to the synergistic interactions among met-

als. Although metal concentrations increased in most

cases, only Pb exceeded recommended level for human

consumption (0.2 Pb mg/L) by 1.1-fold to 2.1-fold.

Keywords Atlantic salmon � Heavy metals �Accumulation � Inland water standard � Maximum-

permissible-amount � Synergistic interactions

Copper, zinc, nickel, chromium, cadmium and lead are

listed as priority hazardous substances (pollutants) in many

countries because of their toxicity, persistence, and affinity

for bioaccumulation (European Parliament and Council

Directive 2000/60/EC; US EPA 2009).

Fish are among the vertebrates having two routes of

metal acquisition: from the water and from the diet (direct

and trophic routes) (Bury et al. 2003).

Most studies into the effects of metals on fish are

addressed to a particular metal. Meanwhile, in the natural

environment, fish are exposed to different metal mixtures,

which are usually more toxic than individual metal as their

action is additive or more-than-additive (synergistic). It

seems that interactions between different metals are related

to their competitive uptake from the environment and to

different distribution in fish tissues, which results from that

certain metals affect the accumulation of other metals in

fish (Jezierska and Witeska 2001).

Although the influence of the presence of particular

metals in the medium on accumulation of others in fish has

been proved experimentally (see Van Hoof and Nauwelaers

1984; Allen 1994; 1995; Pelgrom et al. 1995; Ribeyre et al.

1995.

Ghosh et al. 2007; Palaniappan and Karthikeyan 2009)

little information is compiled on accumulation in fish

exposed to metal mixtures at environmentally realistic

concentrations corresponding to those, which could occur

in an ordinary environment.

The objectives of this study were to (1) determine

experimentally the patterns of accumulation of priority

heavy metals (Cu, Zn, Ni, Cr, Pb, Cd) in different body

tissues of Atlantic salmon (muscle, gills, liver and kidneys)

after 14-day exposure via the direct route either to their

model mixture (HMMM) or to Ni, Cr and Pb singly at a

concentration corresponding to Lithuanian inland water

standards, (2) establish whether metals singly are accu-

mulated quite different than in mixture (3) and whether

metals in mixture at limit concentrations are really safe and

can completely protect aquatic life from harmful effects

from the point of view of metal accumulation in fish body

tissues.

Nickel, Pb and Cr were chosen because according to the

results of monitoring studies of heavy metal accumulation

G. Svecevicius (&) � G. Sauliut _eInstitute of Ecology, Nature Research Centre, Akademijos 2,

08412 Vilnius-21, Lithuania

e-mail: Gintaras.Svecevicius@takas.lt

R. L. Idzelis � J. Grigeleviciut _eDepartment of Environmental Protection, Vilnius Gediminas

Technical University, Saul _etekio 11, 10223 Vilnius, Lithuania

123

Bull Environ Contam Toxicol (2014) 92:440–445

DOI 10.1007/s00128-014-1237-2

in fish and bottom sediments in Lithuania (Project ‘‘Heavy

metal accumulation …’’ 2004), these metals often exceed

the levels recommended for human consumption in fishes

from natural water bodies.

Materials and Methods

The tests were conducted on hatchery-reared one-year-old

Atlantic salmon Salmo salar Linnaeus, 1,758 smolts. The

fish were obtained from M _eskerin _e hatchery (Svencionys

District, Lithuania). Average total length of test fish was

132 ± 3.3 mm, and the total weight was 20 ± 1.5 g

(mean ± SEM, n = 35, respectively). The fish were

acclimated to laboratory conditions for 1 week prior to

testing. They were kept in flow-through 1,000-L holding

tanks supplied with aerated deep-well water (minimum

flow rate 1 L 1 g-1 of their wet body mass per day), under

natural illumination and were fed commercial trout feed

(SKRETTING T-2P Supra) daily in the morning; the total

amount was no less than 1 % of their wet body mass per

day. During the tests, the fish were fed in the same manner.

Deep-well water was used as the dilution water. Average

hardness of the water was approximately 284 (271–296) mg/

L as CaCO3, alkalinity was 200 (190–210) mg/L as CaCO3,

pH ranged from 7.9 to 8.1, temperature was maintained in

10–11�C, dissolved oxygen concentration was maintained at

10–11 mg/L, and dissolved organic carbon (DOC) was less

than the detection limit (\0.3 mg/L).

Reagent grade heavy metal salts («REACHIM» Com-

pany, Russia) were used as the toxicants. Stock solution

was prepared by dissolving necessary amount of the salt in

distilled water, the final concentration being recalculated

according to the amount of heavy metal ion.

The tests were conducted under semi-static conditions

on five groups consisting of seven individuals (four treat-

ments and one control) using glass tanks of 30-L total

volume (20 9 30 9 50 cm in size) filled to a level of 2/3

with continuously aerated dilution water. Test fish were

exposed for the 14-day period to a six heavy metal model

mixture (HMMM) and singly to Ni, Cr and Pb at a con-

centration corresponding to Lithuanian inland water stan-

dards or Maximum-Pemissible-Concentrations (MPC) for

the receiving water-bodies (accepted in 2006 according to

the European Parliament and Council Directive 2000/60/

EC and European Commission recommendation 2006/283/

EC) (Table 1). Test solutions and clean water were

renewed every day, and test fish were transferred into

freshly prepared solutions after they were fed.

After the testing was completed, fish (of control and

metal-exposed groups) were scarified and stored frozen.

Later, they were used in the removal of needed tissues:

muscle without skin (3–5 g), gills (whole organ), liver

(whole organ) and kidneys (whole organ).

The samples were dried in a hot air oven at 85�C for

24 h and digested in aqua regia (concentrated HNO3 and

HCl at a ratio of 1:3 v/v) for 50 min at a temperature

*180�C using microwave digestion system ETHOS

(Milestone, USA). After cooling solutions were filtered

through a 0.45 lm glass filter and diluted with deionized

water up to 50 mL. Metal concentrations were measured by

atomic absorption spectrophotometry on AAS Buck Sci-

entific 210VGP with flame or graphite furnace techniques

in accordance with standardized procedures (LST ISO

11047:2004 en 2004) final concentration being expressed

as mg/kg of wet weight.

The amount of oxygen in the tanks as well as temper-

ature and pH was measured routinely with a hand-held

multi-meter (WTW Multi 340i/SET, Germany). Nominal

heavy metal concentrations were checked during blank

tests (without fish) with an atomic absorption spectropho-

tometer (SHIMADZU AA-6800, Japan) by graphite fur-

nace technique using proprietary software. Each sample

was analyzed in triplicate. Mean measured concentrations

were within 5 %–10 % of the target.

The data were analyzed statistically using two-way

MANOVAs (2- and 3-level, all multivariate tests of sig-

nificance in both cases showed p \ 0.001) followed by

Bonferroni post hoc test through STATISTICA 6.0 (Stat-

Soft Inc., Tulsa, Oklahoma, USA) software.

Results and Dicussion

The data obtained showed that in most cases (17/7) the

amount of heavy metals in body tissues of HMMM-

exposed fish was significantly higher as compared to con-

trol fish (Fig. 1). Maximum exceptions (3) were found in

the kidneys. Heavy metal accumulation order in Atlantic

salmon muscle and gills was as follows: Zn [ Ni [Pb [ Cu [ Cr [ Cd, while in the liver: Zn [ Cu [ Pb [Ni [ Cr [ Cd and in the kidneys: Zn [ Cu [ Pb

[ Cr [ Ni [ Cd.

Table 1 Heavy metals and their test concentrations

Heavy metal Source Maximum-permissible-

concentration (MPC) (mg/L)

Cu CuSO4�5H2O 0.01

Zn ZnSO4�7H2O 0.1

Ni NiSO4�7H2O 0.01

Cr K2Cr2O7 0.01

Cd Cd(CH3COO)2�2H2O 0.005

Pb Pb(NO3)2 0.005

Bull Environ Contam Toxicol (2014) 92:440–445 441

123

Quantitatively maximum levels in the Atlantic sal-

mon body tissues found were of Zn, while the mini-

mum of cadmium. Nickel was in the second place.

Zinc was accumulated mostly in the muscle, at least in

the liver.

Accumulation of Ni, Pb and Cr in half of the cases (6/6)

was significantly influenced by the presence of HMMM in

the water (Fig. 2).

Nickel concentrations in HMMM-exposed fish muscle

and gills were significantly higher than those in Ni-exposed

Fig. 1 Heavy metal

concentration in body tissues of

mixture-exposed (HMMM)

Atlantic salmon: muscle (a),

gills (b) liver (c) and kidneys

(d) (mean ± SD). Asterisks (*)

denote values significantly

different from control

(p B 0.05)

442 Bull Environ Contam Toxicol (2014) 92:440–445

123

fish (by 1.2-fold to 1.9-fold). No significant difference

between Pb concentration in HMMM-exposed and Pb-

exposed fish muscle was found. Lead concentrations in

HMMM-exposed fish gills and kidneys were significantly

higher than those in Pb-exposed fish (by 1.3-fold to 1.4-

fold). No significant difference between Cr concentration

in HMMM-exposed and Cr-exposed fish gills and liver was

found. Chromium concentrations in HMMM-exposed fish

muscle and kidneys were significantly higher than those in

Cr-exposed fish (by 1.3-fold to 1.4-fold). Probably, this

occurs due to the synergistic interactions among the metals.

The data obtained here generally are in agreement with

the results of other studies on metal interaction effects on

their accumulation in fish: Ni ? Cu and Ni ? Cr in roach

Rutilus rutilus (Van Hoof and Nauwelaers 1984); Hg ? Cd

and Hg ? Pb in Oreochromis aureus (Allen 1994);

Cd ? Hg and Cd ? Pb in O. aureus (Allen 1995);

Cu ? Ag ? Se ? Zn ? Hg in zebrafish Brachydanio re-

rio (Ribeyre et al. 1995); Cu ? Cd in tilapia Oreochromis

mossambicus (Pelgrom et al. 1995); Pb ? Cr in Labeo

rohita (Ghosh et al. 2007) and Ni ? Cr in Cirrhinus mri-

gala (Palaniappan and Karthikeyan 2009).

Fig. 2 Comparison of heavy

metal concentration in the

mixture-exposed (HMMM) and

single metal-exposed Atlantic

salmon body tissues: nickel (a),

lead (b) and chromium

(c) (mean ± SD). Asterisks (*)

denote values significantly

different from control, and

grades (#) denote significant

differences between single

metal and mixture-exposed fish

at p B 0.05, respectively

Bull Environ Contam Toxicol (2014) 92:440–445 443

123

In this study, Maximum-Permissible-Amounts (MPA) in

fish indicated in the Lithuanian hygiene standard HN

54:2001: Zn-40, Cu-10, Ni-0.5, Cr-0.3, Pb-0.2 and Cd-

0.05 mg/kg of raw mass, respectively (Project ‘‘Heavy

metal accumulation …’’ 2004) in most cases were not

exceeded, i.e. they were below the recommended levels for

human consumption. Lead was an exception. Its concen-

tration in the gills, liver and muscle exceeded MPA by 1.2,

1.3 and 2.1-fold, in HMMM-exposed fish, and by 1.1 and

2.0-fold in the liver and muscle of Pb-exposed fish,

respectively. It is obvious that here the active uptake of Pb

via the direct route is going on and the presence of other

metals in the water partly promotes it. Apparently, Pb

inland water standard of 0.005 Pb mg/L is too high. We

suppose that it should be revised and decreased at least by

twofold.

Data obtained showed that heavy metal accumulation in

Atlantic salmon was metal and tissue specific, i.e. different

tissues showed a different capacity for accumulating heavy

metals. In general, all tissues contained high concentrations

of Zn but much lower concentrations of Cu, Ni, Cr, Pb, and

Cd. These results coincide with the studies of heavy metal

accumulation in Atlantic salmon in the field. Thus, Yan-

cheva (2010) investigated the heavy metal content in

Atlantic salmon smolts from the Storelva River (Norway)

affected by the mining industry and found Zn concentra-

tions in the gills, liver and kidneys to be the highest as

compared with other metals.

Zinc and Cu are essential metals. The role of essential

metals in fish organism is important as they take part in

metabolic activities. However, in excess essential metals

are potentially toxic, and to maintain metal homeostasis

organisms must tightly coordinate metal acquisition and

excretion (Bury et al. 2003).

It is surprising that elevated amounts of metals were

accumulated in the Atlantic salmon muscle as it is gen-

erally recognized that freshwater fish muscle is not con-

sidered a metal accumulating tissue (Jezierska and

Witeska 2001). We suppose that here could be a couple

of reasons why the Atlantic salmon accumulated the

highest amounts of metals in the muscle. It seems that the

answer is in the test experimental design and fish

behavior. When the smolts were transferred into test

tanks (seven individuals in each) they distributed in the

tank occupying corners and angles lying on the bottom of

the tank. During all 2-week exposure period fish activity

was low. Meanwhile, it is well known that Atlantic sal-

mon is rheophilous species. In the nature salmon are very

active. They continuously demonstrate rheotaxis, actively

search for food and catch it intensively, perform distant

and long-lasting anadromous and catadromous migra-

tions. It seems that if the fish are active they rather

release the metals from the muscle and other tissues, and

field studies confirm it. Thus, Ray (1978) investigated Pb

amounts in Atlantic salmon parr and grilse (one-sea-year)

body tissues from the Miramichi River (Canada) which

was impacted by metal-mining industry. Based on the

data obtained the following bioaccumulation order could

be built: kidney [ liver [ gills [ spine [ muscle. It is

obvious that the muscle is on the last place.

Atlantic salmon is an important commercial and sport

fishing species. Therefore, metal accumulation studies

should be continued. Another experimental design pattern

should be applied. For example, circular test tanks with

rotating water current to induce rheotaxis in test fish could

be tried, etc. Great attention should be paid for the research

into Pb accumulation patterns.

Acknowledgments This work is funded by Research Council of

Lithuania, Project No. MIP-038/2012.

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