Biological responses in edible crab, Callinectes amnicola that couldserve as markers of heavy metals pollution

Adebayo A. Otitoloju Æ Olugbenga K. Elegba ÆAdesola O. Osibona

Published online: 16 July 2008

� Springer Science+Business Media, LLC 2008

Abstract Responses of lagoon crab, Callinectes amni-

cola were explored as useful biological markers of heavy

metal pollution. The toxicity level of the metals based on

the 96-h LC50 values showed that copper with LC50 value

of 0.018 mM was found to be two times more toxic than

Lead (0.041 mM) against the lagoon crab, C. amnicola.

The exposure of the lagoon crab to sublethal concentrations

(1/100th and 1/10th of 96-h LC50 values) of Cu and Pb

compound, respectively, resulted in the bioaccumulation of

the test metals to varying degrees in the selected organs

that were dependent on the type of metal and concentration

of metal compound in the test media. The degree of metal

(Cu and Pb) accumulation was generally in the following

order: gills [ muscle [ heptopancrease. Exposure of the

crabs to sublethal concentrations of the metals also caused

pathological changes such as the disruption of the gill fil-

aments and degeneration of glandular cells with multifocal

areas of calcification in the hepatopancreas. A reduction in

the weight of the exposed animals over a 14-day period of

observation was also recorded. The significance of these

results and the usefulness of the biological endpoints in

monitoring programmes aimed at establishing the total

environmental level of heavy metals in aquatic ecosystems

were discussed.

Keywords Heavy metals � Biomarkers � Histopathology �Bioaccumulation � Biomonitoring

1 Introduction

Real life tragic experiences of heavy metal pollution such

as Minamata and Itai-Itai diseases have shown the devas-

tating effects that the accumulation of heavy metals in

animal/plant tissues which serve as food source could have

on higher predators, particularly man ((Kurdland 1960;

Harada and Smith 1975). As a result, heavy metals have

been categorized as priority pollutants which must be

continually monitored especially in edible organisms even

where they occur at levels below their toxic thresholds in

the ambient environment, since the environmental persis-

tence and non-biodegradable nature, over the years create a

bioconcentration or bioaccumulation in organism that

cannot excrete the absorbed metals (Otitoloju and Don-

Pedro 2002, 2004).

Although relatively high concentrations of metal is

generally required to cause an acute toxic response to

macro-invertebrates, chronic toxicity studies have shown

lethal or sublethal responses at very low levels. Many

essential metals that are required in trace concentrations for

a number of biological processes have been found to

become highly toxic to exposed organisms when presented

over long periods at relatively low concentrations. As a

result, it is pertinent to carry out ecotoxicological studies

on such animals aimed at establishing the toxicity levels of

the changing concentrations, distribution in the various

compartment of the organism and to demonstrate the

effects of the sublethal concentration in the exposed

organisms.

The increasing emphasis on the need for a more com-

plete assessment and monitoring of aquatic ecosystems

other than the mere chemical monitoring of water and

sediment have highlighted the need to deploy appropriate

biological indices for these locations. Bioaccumulation

A. A. Otitoloju (&) � O. K. Elegba

Department of Zoology, Ecotoxicology Laboratory,

University of Lagos, Lagos, Nigeria

e-mail: bayotitoloju@yahoo.com

A. O. Osibona

Department of Marine Sciences, University of Lagos,

Lagos, Nigeria

123

Environmentalist (2009) 29:37–46

DOI 10.1007/s10669-008-9180-6

studies in addition to histopathological biomarkers have

been identified as definite biological endpoints of historical

exposure and thus serve as better indicators of environ-

mental stress (Matthiessen et al. 1993; Stentiford et al.

2003; Stentiford and Feist 2005). The use of biomarkers

may provide important information which once validated

through laboratory studies can provide direct measures of

actual effects of heavy metals upon living organisms in the

field, thereby overcoming large areas of uncertainty

implicit in normal risk assessment (Varanasi et al. 1987;

Vethaak and Jol 1996).

With regards to copper toxicity, Cu was found to be

toxic to aquatic invertebrates such as Typantonous fuscatus

Clibaranus africanus, Sesarma huzardi (Otitoloju and

Don-Pedro 2002). Exposure to Cu has been reported to

cause cellular stress apparent due to reduced lysosomal

stability (Svendsen and Weeks 1997; Viarengo et al. 2000).

Physiological impairment is also manifested in a number of

ways including catabolism, reduction in haemolymph

protein content (Weeks et al. 1993; Lin and Chen 2001),

decrease or increase in heart beat rate (Curtis et al. 2000;

Bamber and Depledge 1997) and induction of methallo-

thionein production in crabs (Brown et al. 2004).

Lead (Pb) is also one of the extensively exploited metals

that has been found in very high concentrations in the

environment. The inclusion of Pb in gasoline as an anti-

knock contribute to its occurrence in the air, which is

transported to the streams and rivers by run-off where fish

and other aquatic organisms take it up and incorporate in

their bodies. The transfer of lead between a primary con-

sumer, the zooplanktonic crustacean Daphnia magna and a

secondary consumer, the fish (Poecilia reticulum) hence

establishing its biomagnification potential and the inherent

public health risk status was also reported (Kakkar and

Jaffery 2005). Exposure to Pb in the environment has been

recognized to be a serious public health problem (WHO

1995) hence its inclusion in this study.

Callinectes amnicola is one of the most common estu-

arine macro-invertebrates and is generally abundant

throughout the Lagos lagoon. Lagoon crabs are a crucial

factor in the brackish water food web because of their

opportunistic feeding habits, high abundance levels and

their great tolerance of salinity. The crabs are being widely

used as food and feed supplement and are considered as an

important shell fishery product. Due to its abundance and

the fact that the crab is being consumed by a large popu-

lation of people especially those living around the coastal

region, the public health risk associated with its con-

sumption need to be monitored continually. and its

usefulness as a biological monitor for assessing the health

status of aquatic ecosystem determined.

In view of the above, the objectives of this study are to

determine the level of some heavy metals accumulated in

various organs of the edible crabs and also establish other

biological endpoints which can serve as useful biomarkers

of metal pollution for assessing the health status of aquatic

ecosystem.

2 Materials and methods

2.1 Test animal

2.1.1 Callinectes amnicola (Crustacean; Decapoda,

Brachyura, Portunidae) (L)

Animals of similar sizes (6–8 cm carapace width and

27–32 g weight) were collected using crab traps and dip

net from the Lagos lagoon. The animals were collected

from the same end of the lagoon in order to reduce vari-

ability in biotype. These were transported as quickly as

possible to the laboratory in baskets from the collection site

and kept in plastic holding tanks (50 cm x 35 cm x 30 cm),

which contained aerated lagoon water (6 l).

Lagoon crabs collected were maintained in the plastic

holding tanks with a maximum of four specimens per tank

to avoid overcrowding. These were shaded with black

polythene to prevent excess light penetration. The animals

were allowed 8 days to acclimatize to laboratory (R.H

70 ± 2%; temperature 27 ± 2�C) and experimental con-

ditions before being exposed to chemicals. During this

period the animals were fed with either Tilapia fingerlings

or commercial fish pellets once daily.

All bioassays were carried out using dechlorinated tap

water. This was achieved by aerating the tap water for

several days. Only inter molt crabs were used and indi-

viduals with mutilated appendages prior to experimentation

were discarded.

2.2 Test chemicals

The heavy metals used for this study were obtained as

metallic salts of Fisons laboratory reagents, analytical

grades of the following types;

(a) Copper as CuSO4 � 5H2O

(b) Lead as Pb(NO3)2

The test chemicals were chosen as they are wide spread

contaminant in the Lagos lagoon (Otitoloju et al. 2007).

2.3 Preparation of test media including application

of toxicants

A pre-determined amount of each heavy metal compound

was weighed out (using an Oertling 30TD analytical bal-

ance) and to this was added a given volume of diluent to

38 Environmentalist (2009) 29:37–46

123

obtain stock solution of known strength. Individual test

solution of copper and lead compound were prepared by

diluting the appropriate volume of stock solution with

dechlorinated water.

2.4 Assessment of mortality

Individual crabs were taken to be dead if no movement of

the body and appendages was observed. This can be further

confirmed by prodding the individual with a glass rod and

those which failed to respond by moving away from initial

position or raised their appendages were considered dead.

2.5 Bioassay

2.5.1 Relative acute toxicity of copper and lead

compounds against Callinectes amnicola

Following the establishment of the range of activity of the

test compounds in the preliminary range finding experi-

ment, the test animals were exposed to several concentrations

of each heavy metal salt and untreated control as follows.

Copper (CuSO4 � 5H2O) against C. amnicola at 0.0001,

0.0003, 0.0006, 0.0013, 0.0063 mM and untreated control.

Lead {Pb(NO3)2} against C. amnicola at 0.0003,

0.0017, 0.0033, 0.005, 0.0067 mM and untreated control.

Eight (8) live crabs of similar sizes were carefully

handpicked at random into the test media in bioassay

container. Four replicates were set up for each treatment

i.e. two crabs were exposed per container for each test

concentration and control.

Mortality assessments were carried out once every 24 h

over a 96 h period.

2.6 Sublethal studies

2.6.1 Bioaccumulation studies

After determining the 96-h LC50 values for each of the two

metals, two sublethal concentrations (1/100th and 1/10th

LC50 values) of each metal and control were prepared. A

total of eight test animals were exposed per sublethal

concentration or control in four replicates i.e. two crabs per

replicate to the metal solution for 14 days in order to

investigate the rate of bioaccumulation of the test metals in

the gills, hepatopancrease and muscle.

Sublethal concentrations under which bioaccumulation

of test metals by C. amnicola was investigated are as

follows:

(a) Cu (CuSO4 � 5H2O) was tested at:

0.0018 mM (1/10th of the 96 h LC50)

0.00018 mM (1/100th of the 96 h LC50)

(b) Pb (Pb(NO3)2 was tested at:

0.0041 mM (1/10th of the 96 h LC50)

0.00041 mM (1/100th of the 96 h LC50)

2.6.2 Determination of concentration of metals

accumulated in selected organs of Callinectes

amnicola

2.6.2.1 Digestion of samples The whole animal samples

(deshelled) were properly cleaned (by copiously rinsing all

exposed and partially exposed parts) with distilled water to

remove debris and other external adherents. The gills,

hepatopancrease and flesh were collected and oven dried at

60�C for 48 h. The dried samples were then grounded

using a mortal and pestle and sieved using a 200 lm to

normalize for particle size. A portion of 2 g from the dried

sample was placed in a glass beaker and digested using a

freshly prepared mixture 1:2 of deionized water and Conc.

HNO3. (FAO/SIDA 1986).

2.6.2.2 AAS determination of heavy metal in samples

(gills, hepatopancrease and flesh) All digestates and

extracts obtained were filtered through Whatman No.1

filter paper and made to mark with deionized water in a

100 cc volumetric flask. The heavy metal concentration in

each digested sample was determined by comparing their

absorbences with those of standards (Solution of known

metal concentrations) using a Perkin Elmer (Precisely) A

Analyst 200 AAS. For data quality, particular attention was

paid to the cleanliness of all wares. In addition, factory

prepared AAS standard solutions were run as samples for

accuracy check after every five measurements.

2.6.3 Histopathological studies

A similar experiment as described above under sublethal

studies was carried out. For these studies, at pre-deter-

mined day intervals (days 0, 7, 14), 2 live C. amnicola per

replicate making four per treatment including control were

randomly selected and dissected. The gills, hepatopan-

crease and flesh were carefully removed and fixed in

Boiun’s fluid pending further histology works. The fixed

samples were transferred to phosphate buffer (pH 6.8),

after 7 h of fixation in Boiun’s fluid. The tissues were then

dehydrated in graded alcohol, cleared in xylene before

embedding in paraffin wax (melting point 56.0�C). Serial

sections of 2–5 lm thickness was cut in rotary microtome

then passed through xylene followed by absolute alcohol

and water. The sections were stained with Haematoxylin

and Eosin, dehydrated in graded alcohol, cleared in more

Environmentalist (2009) 29:37–46 39

123

xylene and mounted in Canada balsam. The slides were left

to dry on the hot plate for 24 h before observation under

the light microscope.

2.6.4 Weight changes experiment

A total of eight test animals were exposed per sublethal

concentration or control to the metal solution for 14 days.

The average weight of the crabs was taken on day 0 and 14

of the experiment with the aid of a top loading compact

weighing balance.

For all the sublethal experiments, a semi-static bioassay

procedure was adopted to avoid drastic changes in con-

centration of test media evaporation and excessive

reduction in dissolved oxygen level. In this semi-static

procedure, each test media was changed into a fresh

solution once every 3 days, to exactly the same concen-

tration of heavy metal salt or untreated control. The same

exposed test animals were transferred into the freshly

prepared test media over a 14 days period of experimen-

tation. During these experiments, the animals were fed with

periwinkle flesh.

2.7 Statistical analysis

Toxicological dose-response data involving quantal

response (mortality) were analyzed by probit analysis

(Finney 1971). This was done using a computer-based

programme designed by Ge Le Pattourel, Imperial College,

London, as adopted by Don-Pedro (1989). Indices of tox-

icity measurement from this analysis were:

LC50 = Median lethal concentration that causes 50%

response (mortality) of exposed organism.

LC95 = Lethal concentration that causes 95% (mortal-

ity) of exposed organism.

TF = Toxicity factor for relative potency measurement,

e.g. 96-h LC50 of Lead/96-h LC50 of Copper tested

against C. amnicola.

Bioaccumulation factor (BAF) was also estimated as the

ratio of the concentration of the metal ion in animal organ

after 14 days of exposure to the concentration of metals

exposed to in the test media.

Bioaccumulation factor BAFð Þ steady state � 14 daysf g

¼ Concentrationofmetalioninselectedorgan

Concentration of metal ion in test media

3 Results

3.1 Physico-chemical conditions in bioassays during

toxicity testing

The results of the physico-chemical parameters of the test

media during the period of experimentation reveals that the

dissolved oxygen level ranged from 8.30 mg/l (at the

beginning of the experiment) to 6.69 mg/l (before change

to a clean media). The pH and salinity of the test media

remained fairly constant with values ranging from 6.9 to

7.2, and 3% to 5%, respectively. The conductivity and

total dissolved solids in the test media increased from

1.69mS/cm and 0.84ppt to 2.32mS/cm and 1.16ppt,

respectively, over the period of observation.

3.2 Relative acute toxicity of heavy metal salts acting

against C. amnicola

On the basis of the 96-h LC50, CuSO4 (0.018 mM) was

found to be more toxic than Pb(NO3)2 (0.041 mM) tested

against C. amnicola (Table 1). The computed toxicity

factor, based on ratios of 96-h LC50 revealed that Cu was

two times more toxic than Pb when tested against the

lagoon crab. The log-dose probit response plot, depicting

the toxicity profile of copper and lead against C. amnicola

were non-parallel, indicating that comparison of the tox-

icity test chemical are only valid for a level of dose and

response.

3.3 Sublethal studies

3.3.1 Bioaccumulation of Cu ions in selected organs of

Callinectes amnicola

3.3.1.1 Gills Post-treatment analysis of the gills showed

that the animals exposed to sublethal concentrations

Table 1 Relative acute toxicity of CuSO4 and Pb(NO3)2 salts against Callinectes amnicola (Lagoon crab)

Treatment Time (h) LC50 (50% C.L mM) LC95 (95% C.L mM) Slope ± S.E Probit line equation D.F T.F

CuSO4 96 0.018 (0.145–0.002) 2.348 (1.232–4.267) 0.779 ± 0.624 Y = 6.361 + 0.779x 2 1

Pb(NO3)2 96 0.041 (0.052–0.032) 0.073 (0.122–0.044) 6.566 ± 0.624 Y = 14.117 + 0.779x 1 2.27

C.L = Confidence Limit

D.F = Degree of Freedom

T.F = Toxicity Factor

S.E = Standard Error

40 Environmentalist (2009) 29:37–46

123

(0.00018 mM and 0.0018 mM) of the copper ions, accu-

mulated measurable quantities of the metal that were

approximately two times above the levels accumulated by

the animals in untreated control media. Furthermore, at the

end of the 14 days exposure period, there was an overall

net gain of 0.219 mg/l and 0.378 mg/l of Cu over the

respective initial amount of the metal in the gills exposed

to the sublethal concentrations 0.00018 mM (1/100th

LC50) and 0.0018 mM (1/10th LC50), respectively

(Table 2). Comparisons of the concentration of Cu ions

accumulated in the gills exposed to sublethal concentra-

tions, 1/100th and 1/10th LC50 of 96-h LC50 to that of

concentration in the test media revealed that the level

accumulated in the organ were found to be 11.8 and 1.533

times, respectively, higher than the level in the test media

(Table 4).

3.4 Hepatopancreas

Post-treatment analysis of the hepatopancreas revealed that

the level of Cu accumulated in the hepatopancreas of

exposed animals were similar to the level accumulated in

control animals. At the end of the exposure period, there

was an overall net gain of 0.094 and 0.127 mg/l for animals

exposed to sublethal concentration of 0.00018 mM

(1/100th LC50) and 0.0018 mM (1/10th LC50), respectively

(Table 2). Comparisons of the concentration of Cu ions

accumulated in the hepatopancreas exposed to sublethal

concentrations, 1/100th and 1/10th LC50 of 96-h LC50 to

that of concentration in the test media revealed that the

level accumulated in the organ were found to be 26.02 and

2.676 times, respectively, higher than the level in the test

media (Table 5).

Table 2 Accumulation of

heavy metal [Cu] ion by

C. amnicola exposed to

sublethal concentrations

of the metal under laboratory

conditions

* Overall net

gain = Concentration in animal

after 14 days - Concentration

in organ at zero daya 1/100th 96-h LC50 = Values

of the metals ion in the test

compoundsb 1/10th 96-h LC50 = Values

of the metals ions in the

compounds

Treatments (mM) Mean concentration of copper in selected organs

(mg/l wet weight basis)

Overall net gain*

0 day 14 days

Gills

Untreated control 0.312 0.322

0.00018a [1/100th LC50] 0.312 0.531 0.219

0.0018b [1/10th LC50] 0.312 0.690 0.378

Hepatopancrease

Untreated control 1.077 1.084

0.00018a [1/100th LC50] 1.077 1.171 0.094

0.0018b [1/10th LC50] 1.077 1.204 0.127

Muscle

Untreated control 1.276 1.297

0.00018a [1/100th LC50] 1.276 0.516 –0.760

0.0018b [1/10th LC50] 1.276 1.506 0.230

Table 3 Accumulation of

heavy metal [Pb] ion by C.amnicola exposed to sublethal

concentrations of the metal

under laboratory conditions

* Overall net

gain = Concentration in animal

after 14 days - Concentration

in organ at zero daya 1/100th 96-h LC50 = Values

of the metals ion in the test

compoundsb 1/10th 96-h LC50 = Values

of the metals ions in the

compounds

Treatments (mMl) Mean concentration of lead in different organs

(mg/l wet weight basis)

Overall net gain*

0 day 14 days

Gills

Untreated control 0.356 0.420

0.00041a [1/100th LC50] 0.356 0.813 0.457

0.0041b [1/10th LC50] 0.356 1.843 1.487

Hepatopancrease

Untreated control 0.382 0.436

0.00041a [1/100th LC50] 0.382 0.352 –0.03

0.0041b [1/10th LC50] 0.382 0.615 0.233

Muscle

Untreated control 0.327 0.363

0.00041a [1/100th LC50] 0.327 0.949 0.622

0.0041b [1/10th LC50] 0.327 0.849 0.522

Environmentalist (2009) 29:37–46 41

123

3.4.1 Muscle

Post-treatment analysis of the muscle revealed that the

level of Cu accumulated in the muscle of exposed animals

were similar to the level accumulated in control animals.

At the end of the exposure period, there was an overall net

gain of -0.760 mg/l and 0.230 mg/l for animals exposed

to sublethal concentration of 0.00018 mM (1/100th LC50)

and 0.0018 mM (1/10th LC50), respectively (Table 2).

Comparisons of the concentration of Cu ions accumulated

in the muscles exposed to sublethal concentration, 1/100th

and 1/10th LC50 of 96-h LC50 to that of concentration in

the test media revealed that the level accumulated in the

organ were found to be 11.47 and 3.347 times, respec-

tively, higher than the level in the test media (Table 6).

3.4.2 Bioaccumulation of Pb ions in selected organs

of Callinectes amnicola

3.4.2.1 Gills Post-treatment analysis of gills of C. am-

nicola showed that the animals exposed to sublethal

concentration (0.00041 mM and 0.0041 mM) of the lead

compound accumulated measurable quantities of the metal

that were approximately 2–4 times higher than the level

accumulated by the animals in the untreated control media.

Furthermore, at the end of the 14-day exposure period,

there was an overall net gain of 0.488 and 1.487 mg/l of Pb

over the respective initial amount of the metal in the gills

exposed to the sublethal concentrations of 0.00041 mM

(1/100th LC50) and 0.0041 mM (1/10th LC50), respectively

(Table 3). Comparisons of the concentrations of Pb ions

accumulated in the gills of test animals exposed to suble-

thal concentrations, 1/100th and 1/10th LC50 of 96-h LC50

to that of concentration in the test media revealed that the

level accumulated in the organ were found to be 0.6 and 0.1

times, respectively, lower than the level in the test media

(Table 4).

3.4.2.2 Hepatopancreas Post-treatment analysis of the

hepatopancreas showed that the lagoon crabs exposed to

sublethal concentration (0.00041 mM and 0.0041 mM) of

the lead compound accumulated measurable quantities of the

metal that were 1.5 times higher than the levels accumulated

by animals in untreated control media. Furthermore, at the

end of the 14-day exposure period, there was an overall net

gain of -0.03 and 0.233 mg/l for animals exposed to sub-

lethal concentrations of 0.00041 mM (1/100th LC50) and

0.0041 mM (1/10th LC50), respectively (Table 3). Com-

parisons of the concentrations of Pb ions accumulated in the

hepatopancreas of test animals exposed to sublethal con-

centrations, 1/100th and 1/10th LC50 of 96-h LC50 to that of

concentrations in the test media revealed that the level

accumulated in the organ were found to be 0.3 and 0.1 times

lower than level in the test media (Table 5).

3.4.2.3 Muscles Post-treatment analysis of the muscles

revealed that the levels of Pb accumulated in the muscle of

exposed animals were approximately three times above the

level accumulated by animals in untreated control media.

At the end of the exposure period, there was an overall net

gain of 0.622 mg/l and 0.522 mg/l of Pb for animals

exposed to sublethal concentrations of 0.00041 mM

(1/100th LC50) and 0.0041 mM (1/10th LC50), respectively

Table 4 Bioaccumulation factor in relation to level of heavy metals

[Cu and Pb] accumulated in gills of C. amnicola exposed to sublethal

concentrations of the metals under laboratory condition

Treatment

metals

Mean concentration

of metals in test

media (mg/l)

Mean concentration

of metals in organs

after 14-days

(mg/kg)

BAF*

Control

Cu 0.312 0.322

Pb 0.356 0.420

1/100th of 96-h LC50

Cu (0.00018) 0.045 0.531 11.80

Pb (0.00041) 1.357 0.813 0.599

1/10th of 96-h LC50

Cu (0.0018) 0.450 0.690 1.533

Pb (0.0041) 13.571 1.843 0.136

* BAF ¼ Bioaccumulation factor

¼ Concentration of metal ion in selected organ

Concentrations of metal ion in test media

Table 5 Bioaccumulation factor in relation level of heavy metals

[Cu and Pb] accumulated in hepatopancreas of C. amnicola exposed

to sublethal concentrations of the metals under laboratory condition

Treatment

metals

Mean concentration

of metals in test

media (mg/l)

Mean concentration

of metals in organs

after 14-days

(mg/kg)

BAF*

Control

Cu 1.077 1.084

Pb 0.382 0.436

1/100th of 96-h LC50

Cu (0.00018) 0.045 1.171 26.02

Pb (0.00041) 1.357 0.352 0.259

1/10th of 96-h LC50

Cu (0.0018) 0.450 1.204 2.676

Pb (0.0041) 13.571 0.615 0.045

* BAF ¼ Bioaccumulation factor

¼ Concentration of metal ion in selected organ

Concentrations of metal ion in test media

42 Environmentalist (2009) 29:37–46

123

(Table 3). Comparisons of the concentrations of Pb ions

accumulated in the muscles of test animals exposed to

sublethal concentrations, 1/100th and 1/10th LC50 of 96-h

LC50 to that of concentrations in the test media revealed

that the levels accumulated in the organ were found to be

0.70 and 0.06 times, respectively, lower than the level in

the test media (Table 6).

3.4.3 Hispathological studies

The results of the histopathological effects of Cu and Pb on

the gills and hepatopancreas are shown in Figs. 1 and 2.

The results show that there were various degrees of

histological alterations observed in the organs examined

(gills—Fig. 1a, b and Hepatopancreas—Fig. 2a, b). Expo-

sure of lagoon crab to sublethal concentrations of Pb in

particular was found to result in the disruption of the gill

filaments (Fig. 1b) accompanied by precipitation of Pb

granules (Fig. 1b). Additionally, sections of crab’s hepa-

topancreas exposed to Pb compounds also revealed a

general degeneration of glandular cells with multifocal

areas of calcification in the hepatopancreas.

3.4.4 Weight changes experiment

Results of the weight changes in C. amnicola exposed to

sublethal concentrations of Cu salt showed that the test

animals lost about 12.5–19.05% of its weight over the

14 days period of observation. On the otherhand lagoon

crabs exposed to sublethal concentrations of Pb lost about

10.5–12.9% of the body weight over the period of observa-

tion. Animals in control media however gained about 10% of

the body weight during the period of observation (Fig. 3).

4 Discussion

In this study, the differential toxicity of two metallic salts;

CuSO4 and Pb(NO2)3 against C. amnicola was demon-

strated. From the established toxicity result, CuSO4 (96-h

LC50 value of 0.018 mM) was found to be two times more

toxic than Pb(NO2)3 (96-h LC50 0.041 mM) when acting

singly against the lagoon crab, C. amnicola. This result

agrees with those of several authors including Otitoloju and

Don-Pedro (2002) who reported the general toxicity rank-

ing order of heavy metals on benthic animals of the Lagos

Table 6 Bioaccumulation factor in relation level of heavy metals [Cu

and Pb] accumulated in muscles of C. amnicola exposed to sublethal

concentrations of the metals under laboratory condition

Treatment metals Mean concentration

of metals in test

media (mg/l)

Mean concentration

of metals in organs

after 14-days (mg/l)

BAF*

Control

Cu 1.276 1.297

Pb 0.327 0.363

1/100th of 96-h LC50

Cu (0.00018) 0.045 0.516 11.47

Pb (0.00041) 1.357 0.949 0.716

1/10th of 96-h LC50

Cu (0.0018) 0.450 1.506 3.347

Pb (0.0041) 13.571 0.849 0.063

* BAF ¼ Bioaccumulation factor

¼ Concentration of metal ion in selected organ

Concentrations of metal ion in test media

Fig. 1 Gills Filament of

Callinectes amnicola. (a)

Normal section of gills for

control animals. (b) Disrupted

gill filaments (black arrows) in

crabs exposed to sublethal doses

of Pb compound accompanied

by precipitation of Pb granules

(white arrow)

Environmentalist (2009) 29:37–46 43

123

lagoon as follows: Hg [ Cd [ Cu [ Zn [ Pb. Clark

(1992), also reported the same order of toxicity with

stickleback. The observation of increased concentration of

these metals in single action laboratory studies has also

been well established by Bryan and Langston 1992, Baron

1995 and Oyewo 1998.

The observed toxicity of different heavy metals can be

attributed to several factors such as metal compound tested,

solubility of salts, predominant ions in test solution, and

physico-chemical characteristics of the test solution and the

mechanism of action of the different metals. Other factors

which may affect metal’s toxicity or susceptibility of test

animals include the formation of complexes with protein,

e.g. metallothionein complexes formation of encapsulated

metal granules, metabolism and excretability. The low

toxicity nature of Pb compound may be because of the free

inorganic ion Pb2+ which is usually dominant in Pb is not

lipid soluble, hence transfer across membranes may be

inhibited (Otitoloju 2002). This study also revealed that Cu

is more toxic than Pb to aquatic macro-invertebrates.

Hence, their introduction into the aquatic environment via

industrial effluent must be minimized through effective

control/management strategies and where possible Pb

should be used in place of copper.

The level of the metals accumulated in the organs of the

test animal was found to be higher in animals exposed to

1/10th LC50 than in those exposed to 1/100th LC50. The

concentrations in exposed animals were also generally

higher than the concentrations detected in control animals.

This observation therefore implies that the concentration of

metals accumulated was directly related to the concentra-

tion of the metal present in their environment. This result

of metal uptake is in agreement with studies of Chukwu

(1991) and Oyewo (1998) who demonstrated that the

concentration of metals in exposed animals such as Pal-

aemonetes africanus, Tympanotonous fuscatus, Tilapia

guieenensis and Clibaranus africanus increased with time

of exposure and test medium concentration.

On the basis of the concentration of metals accumulated

in the different organs, the order of metal accumulation in

the organs is as follows: gills [ muscle [ hepatopan-

crease. This result is somewhat similar to the result of

Radhakrishnaiah (1988) who reported the order of Cu

accumulation in freshwater fish Labeo rohita (Hamlton) as

gill [ brain [ muscle [ liver. According to Otitoloju and

Don-Pedro (2006), the bioaccumulation of heavy metals in

animal tissues occurs as a result of competing rates of

chemical uptake and excretion. Accumulation of metals

will therefore occur when the rate of uptake is higher than

rate of excretion. Furthermore, the synthesis of low

molecular weight proteins e.g. metallothionein which form

complexes with the metal ion, as well as the formation of

exposed animals have also been reported to be responsible

Fig. 2 Hepatopancreas of

Callinectes amnicola. (a)

Normal section of

hepatopancreas showing distinct

hepatocytes (H) in control

animals. (b) Degenerated

glandular cells with multifocal

areas of calcification (arrows) in

crabs exposed to sublethal doses

of Pb compound

-25

-20

-15

-10

-5

0

5

10

15

Cu 1/100 LC50 Cu 1/10 LC50 Pb 1/100 LC50 Pb 1/10 LC50Control

% W

eigh

t Gai

n

Concentrations

Fig. 3 Weight changes in Callinectes amnicola exposed to sublethal

concentrations of heavy metal (Cu and Pb) compounds over a 14-day

observation period

44 Environmentalist (2009) 29:37–46

123

for metal accumulation in some aquatic mollusc (Langston

and Zhou 1987; Clark 1992).

The detection of higher concentration of Cu in the gills

could be as a result of Cu being a component of haemocyanin

which is a respiratory pigment with its site of action at the

gills. Also the fact that the gills are in direct contact with the

surrounding water may account for the higher level of copper

accumulation in the organ. The detection of lower concen-

tration of Cu and Pb ions in the hepatopancrease of the

Lagoon crab exposed to sublethal concentrations indicates

that this organ may be the primary organ for metal detoxi-

fication and excretion.

Generally, a higher concentration of Pb was accumulated

in the different organs of the crab than Cu. In fact the con-

centration of Cu accumulated in the organs were found to

fluctuate significantly over the observation period. Therefore

suggestive that the test animal may have capacity to

metabolize and excrete its body burden of Cu over time. The

ability of aquatic invertebrates such as Tympanotonus fusc-

atus to excrete rapidly its body burden of Cu has also been

reported by Otitoloju and Don-Pedro (2002). With regards to

lead however, the concentration of lead accumulated

increased steadily with time of exposure. Therefore indi-

cating that the animal may not have the potential to excrete

the metal ions from its body, but rather have the capacity to

store it up in a harmless state e.g. by storing the metal within

membrane in the cytoplasm or secretion of low molecular

weight proteins which binds the metals. The particular

mechanism(s) by which T. fuscatus accumulate Pb in its

body tissues should therefore merit further investigation.

This study also showed that the exposure of C. amnicola

to sublethal concentration of Cu and Pb caused a marked

decrease in weight of the exposed animals compared to

control organisms. The weight loss was also found to be

more pronounced in crabs exposed to Cu than Pb exposed

ones. The loss of weight in the exposed animals may be

attributable to the observed reduction in locomotory and

feeding activities in the exposed animals.

In conclusion, the range of biological responses

observed in this study such as the ability of lagoon crab

to accumulate heavy metals to levels that are several

folds higher than the environment, the toxicopathic tissue

alterations from the histopathological studies and the

remarkable weight loss in exposed crabs represent impor-

tant biological endpoints of contaminant exposure which

reinforces the inclusion of biological responses in moni-

toring anthropogenic contamination of aquatic ecosystem.

The combination of these biological endpoints with phys-

ical/chemical parameters will provide a clear picture of the

total environmental quality and provide environmental

managers with the necessary tools to make important

decisions that will prevent such cases of metal pollution

resulting in human death.

Acknowledgement The authors are grateful to Dr. J.K. Saliu for his

useful suggestions in the preparation of the manuscript.

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