Toxicity, Feeding Rate and Growth Rate Response to Sub-lethal

Concentrations of Anthracene and Benzo [a] Pyrene in Milksh

Chanos chanos (Forskkal)
L. Palanikumar

A. K. Kumaraguru

C. M. Ramakritinan

M. Anand
Received: 19 July 2012 / Accepted: 10 November 2012 / Published online: 30 November 2012
Springer Science+Business Media New York 2012
Abstract The feeding rate, growth rate and gross con-
version efciency were studied in milksh Chanos chanos
for 28 days of exposure to sub-lethal concentrations of
anthracene (1.00, 2.00, 3.00, 6.00 and 12.0 lg l
-1
) and
benzo [a] pyrene (0.30, 0.70, 1.40, 2.80 and 5.60 lg l
-1
)
under continuous ow through bioassays. Based on
survival and growth data, No Observed Effect Concentra-
tion; Lowest Observed Effect Concentration were esti-
mated after 28 days, the values for anthracene were 2.03
and 3.09 lg l
-1
, and the values for benzo [a] pyrene were
0.82 and 1.46 lg l
-1
, respectively. Anthracene and benzo
[a] pyrene exposure caused reduction in feeding and
growth rate.
Keywords C. chanos Sub-lethal toxicity Feeding rate
Growth rate NOEC LOEC
Coastal environments are subjected to several forms of
disturbance, amongst which chemical pollution associated
with industrial production and high levels of urbanization
are both of major concern (Fernandez et al. 2010). Crude
oil spills, renery activities and industrial and urban wastes
are important sources of polycyclic aromatic hydrocarbons
(PAHs) in aquatic ecosystems (Fernandez et al. 2010).
Polycyclic aromatic hydrocarbons and their halogenated
forms are chemically stable, and due to their lipophilic
nature they can easily penetrate biological membranes and
accumulate in organisms. PAHs are important environ-
mental pollutants because of their ubiquitous presence and
carcinogenicity (Palanikumar et al. 2012a, b). Anthracene
and benzo [a] pyrene is considered an indicator of such
contamination because they usually occur in mixtures of
PAHs (Kim et al. 2008). Marine sh readily take up lipo-
philic organic contaminants such as benzo [a] pyrene from
the marine environment, with a variety of physiological
effects (Kim et al. 2008).
Fishes exposed to PAHs in water column throughout
their life cycle can serve as natural indicators of PAH
contamination in surface waters (Logan 2007). Studies on
shes exposed to sub-lethal environmental stressors prove
to be a sensitive method for predicting the effects on
survival and growth (Cao et al. 2010). Feed intake,
energy metabolism and growth are subject to a complex
interaction of body weight and temperature (Rollo et al.
2006).
Milksh Chanos chanos (Forsskal, 1775) is an impor-
tant tropical marine sh and is cultured in the Philippines,
Indonesia, India and Taiwan producing about 330,000
tonnes every year (Rabanal 1988). The milksh has been
the subject of numerous studies of varied extent and depth
by investigators in relation to biology, aquaculture, hatch-
ery context and toxicity (Bagarinao 1994; Palanikumar
et al. 2012a, b). In view of the above, the present investi-
gations were carried out to assess toxicity, feeding rate,
growth rate and gross conversion efciency rates of
anthracene and benzo [a] pyrene on ngerlings of the
milksh C. chanos (Forsskal) of the Gulf of Mannar,
Southeast coast of India.
L. Palanikumar (&) A. K. Kumaraguru
C. M. Ramakritinan M. Anand
Department of Marine and Coastal Studies, School of Energy,
Environment and Natural Resources, Madurai Kamaraj
University, Madurai 625021, India
e-mail: [email protected]
Present Address:
L. Palanikumar
Crystal Growth Centre, Anna University, Chennai 600025, India
1 3
Bull Environ Contam Toxicol (2013) 90:6068
DOI 10.1007/s00128-012-0895-1
Materials and Methods
Healthy specimens of an milksh C. chanos (Forskkal)
(average size and weight 2.55 0.05 cm; 1.50 0.15 gm)
were collected from Kundukal and Chinnapalam regions
(Latitude 09816.26
0
N and Longitude 079812.88
0
E) of Pam-
ban coast, Gulf of Mannar, Southeast coast of India. Initial
disinfection treatment was carried out using benzyl konium
chloride (1 mg l
-1
) for 1 h and KMnO
4
solution (1 mg l
-1
)
for 1 h and then healthy individuals were separated, accli-
matized for 10 days in large glass aquaria containing aged,
ltered seawater (calcium hardness 395.5 mg l
-1
, magne-
sium hardness 1,410.0 mg l
-1
, dissolved oxygen 5.68
mg l
-1
, in-organic phosphate 3.76 lg l
-1
, nitrite-nitrogen
1.95 lmol l
-1
, nitrate-nitrogen 4.65 mmol l
-1
and
ammonia 0.09 lg l
-1
). Gentle aeration was provided using
oil free air compressor (Elgi Make, Dr. Vayu Air Com-
pressor, Coimbatore, India) supplying air through dispos-
able pasture pipettes connected via plastic tubes. During this
period, the shes were fed on live brine shrimp (Artemia sp.)
nauplii (Palanikumar et al. 2012a). The study has been
approved by Institutional Ethical Committee in accordance
with the ethical standards provided by the Department of
Marine and Coastal Studies, Madurai Kamaraj University,
Madurai, India.
Clean and clear seawater was collected from
Pudhumadam coast, Gulf of Mannar (Latitude 09816.39
0
N
and Longitude 079800.50
0
E) at a distance where 10 m
depth existed. The collected water was stored in settling
tanks of 5,000 l capacity for a period of 15 days. This
conditioned seawater was pumped into a main storage tank
for ltration. From this storage tank, seawater was passed
through sand lter for purication. Then, it was passed
through a 10, 1 l and carbon cartridge lters and nally
through an UV sterilizer. This ltered seawater was used
for the test animals, live feed culture and bioassay tests.
The physico-chemical characteristics of diluted seawater
were analyzed using standard protocols (Grasshoff et al.
1999). The lighting period was maintained at a ratio of
16 h: 8 h day: night period, respectively.
The tests were conducted for a period of 28 days
(Environment Canada EPS 1/RM/44). Test chambers were
maintained in quadruplicate for each test concentration.
Each test chambers contained 10 sh. A total of 40 sh
were used for each concentration. Artemia nauplii were
given as feed for sh, ad libitum until 48 h prior to 28 days
when food was withheld (Gundersen et al. 1996). Feed was
dried in a hot air oven for 24 h to weight constancy and the
water content of the feed was estimated. Dead animals if
any were removed immediately after observation. The ow
through toxicity system was built to supply a constant ow
of seawater and toxicant under test. All tubings used are
exible silicone tubing (Tygon, USA) with specic
dimensions (Tubing ID 3.17 mm). In the ow through
toxicity tests, 20 ml min
-1
seawater control, 0.05 % ace-
tone control and toxicant medium) delivered to test
chamber providing 95 % molecular volume replacement
time of about 30 min (Sprague 1969) with Microprocessor
based peristaltic pumps (Model 1256, Ismatec, USA). The
unfed remains were carefully collected at the outlet using a
net (100 lm) of the test chambers without causing distur-
bance to the sh.
The acute toxicity bioassays procedure based on stan-
dard methods (Sprague 1973; OECD 1993; APHA et al.
1998) was conducted to determine the LC
50
values of
anthracene (CAS no 120-12-1) and benzo [a] pyrene (CAS
no 50-32-8) (Sigma-Aldrich Co, USA). Based on acute
toxicity test results, sub-lethal concentrations were selected
by calculating 1/10th of the LC
50
(96 h) value of anthra-
cene and benzo [a] pyrene (Palanikumar et al. 2012a) and it
was 1.00, 2.00, 3.00, 6.00 and 12.0 lg l
-1
for anthracene
and 0.30, 0.70, 1.40, 2.80 and 5.60 lg l
-1
for benzo
[a] pyrene. The concentrations dissolved in test medium
were estimated thrice at an interval of 7 days with pooled
samples (Fig. 1). The samples were consecutively extrac-
ted with 50 ml of hexane and 25 ml of dichloromethane.
The organic extracts were combined, dried over anhydrous.
Na
2
SO
4
, rotary-evaporated and fractionated in a glass
column lled with neutral alumina (1 g) and silica gel
(1 g), both 5 % water deactivated. Two fractions were
Fig. 1 GC-MS-SIS
chromatogram obtained from
test medium for anthracene
(0.92 lg l
-1
) and benzo
[a] pyrene (0.29 lg l
-1
)
Bull Environ Contam Toxicol (2013) 90:6068 61
1 3
eluted, the rst one with 2 ml of hexane which contained
the aliphatic compounds and the second with 10 ml of
dichloromethane: hexane (30:70 v/v) which contained the
PAHs.
The second fraction was analyzed by Capillary Gas
ChromatographyHigh Resolution Mass Spectrometry
(GC/MS) system of Shimadzu GC/MS 2010 gas chro-
matograph. Separation took place in a DB-XLB column
(60 m 9 0.25 mm 9 0.25 lm) (Agilent, Wilmington),
temperature programmed from 50C (4 min) to 200C at
6C min
-1
and nally to 325C at 4C min
-1
, holding this
temperature for 10 min (Gonzalez et al. 2006). Shimadzu
data system was used for PAH analysis. The recoveries of
spiked PAHs standards ranged from 60 % to 108 %. The
range of concentrations present in test medium was 0.92,
2.03, 3.09, 5.98 and 11.17 lg l
-1
for anthracene and 0.29,
0.82, 1.46, 2.80 and 5.60 lg l
-1
for benzo[a]pyrene.
0.05 % acetone and seawater was maintained as solvent
control and negative control (Table 1). In control medium,
concentrations of anthracene and benzo [a] pyrene was nil.
After 14 and 28 days of sub-lethal experiments, feeding
rate was determined by subtracting the dry weight of the
uneaten food from the dry weight of that given and
expressed as mg dry food consumed per g live sh per day
(Felistarani et al. 1998). The estimated moisture content
(AOAC Method 960.18) of 52 % 1.2 % is reduced from
the initial live weight of the C. chanos for calculating the
feeding rate and growth rate.
Feeding rate
total food consumed mg
no: of days initial live weight of fish g
After 14 and 28 days of sub-lethal experiments, the test
sh were starved for a day to ensure complete evacuation
of gut. They were weighed alive and sacriced. The
sacriced sh were dried to constant weight at 80C.
The dry weight of the individual sh was calculated. The
growth rate or conversion rate, i.e. production was
expressed as mg dry substance gained per gram live sh
per day (Felistarani et al. 1998).
Growth rate

final weight of fish g initial weight of fish g

No: of days initial weight of fish g
After 14 and 28 days of sub-lethal experiments, gross
conversion efciency was calculated as percentage of food
converted into body tissues in relation to feeding rate
(Felistarani et al. 1998).
Gross conversion efficiency
growth rate
feeding rate
100
Linear regression analysis for concentration versus (%)
survival in sub-lethal tests for C. chanos was carried out
using Excel in MS ofce 2007. The sub-lethal toxicity test
estimates are given in terms of two endpoints; the No
Observed Effect Concentration and the Lowest Observed
Effect Concentration were computed by using Dunnett
analysis (USEPA 1994) with Graph Pad prism version 5.0
software. One way analysis of variance (ANOVA) was
used to compare the signicance difference between
control and the various sub-lethal concentrations for
feeding rate, growth rate and gross conversion efciency.
The two way analysis of variance was used to compare the
signicant difference between control and the various
treated concentrations of anthracene and benzo [a] pyrene
for survival, feeding rate, growth rate and gross conversion
efciency. The level of signicance chosen was 0.05,
unless otherwise noted.
Results
Acetone (0.05 %) was used as a solvent in the present
study. Control groups received equal volume of acetone.
The physico-chemical characteristics of test medium were
Table 1 Sub-lethal concentrations of anthracene and benzo [a] pyrene given to C. chanos (n = 3)
Toxicanst Concentration (lg l
-1
)
Anthracene
Nominal Control Solvent control (0.05 %
acetone)
1.00 2.00 3.00 6.00 12.00
Measured (n = 3;
mean SD)
ND ND 0.92 0.05 2.03 0.09 3.09 0.15 5.98 0.14 11.17 1.22
Benzo [a] Pyrene
Nominal Control Solvent control (0.05 %
acetone)
0.30 0.70 1.40 2.80 5.60
Measured (n = 3;
Mean SD)
ND ND 0.29 0.07 0.82 0.11 1.46 0.21 2.57 0.20 4.73 1.20
ND not detected; the minimum detection limit is 10 ng ml
-1
62 Bull Environ Contam Toxicol (2013) 90:6068
1 3
temperature 27 2C, dissolved oxygen 5.58 0.3 mg l
-1
,
salinity 32 1 % and pH 8.2 0.3.
Non-locomotor and locomotor response in swimming
behavior of sh was observed. More immobilized response
was observed in higher concentrations of anthracene and
benzo [a] pyrene when compared to lower concentrations.
At maximum exposure duration of 28 days, sh showed
disorientation of swimming activity and resting sideways in
higher concentrations of anthracene and benzo [a] pyrene.
Survival data of sh exposed to different concentrations
of anthracene and benzo [a] pyrene are presented in Fig. 2a
and b. Survival of 100 % was noticed in control, solvent
control and up to 5.98 lg l
-1
anthracene after an exposure
of 14 days. However, it was 97 % survival in the highest
sub-lethal concentration of anthracene i.e., 11.17 lg l
-1
after 14 days. Survival of 100 % was noticed in sh
exposed to control, solvent control, 0.92 and 2.03 lg l
-1
anthracene in 28 days exposure. In benzo [a] pyrene
treatment, survival of 100 % was noticed in control, sol-
vent control and up to 2.57 lg l
-1
benzo [a] pyrene
exposed sh after 14 days. However, 98 % survival was
observed in the highest sub-lethal concentration of benzo
[a] pyrene. Survival of 100 % was noticed in sh exposed
to control, solvent control and up to 0.82 lg l
-1
benzo
[a] pyrene after 28 days. However, it was 90, 83 and 78 %
in sh exposed to 1.46, 2.57 and 4.73 lg l
-1
respectively.
In lower concentrations, 100 % survival was noticed
after 28 days of exposure in both the toxicants tested and in
higher concentrations survival was more in sh exposed
to anthracene. However, survival decreased with increas-
ing sub-lethal concentrations of anthracene and benzo
[a] pyrene. Based on survival data, No Observed Effect
Concentration (NOEC) and Lowest Observed Effect
Concentration (LOEC) were estimated after 28 days. For
Fig. 2 a Survival of C. chanos versus sub-lethal concentrations of
anthracene after 0 h, 14 and 28 days. Values are mean SD (n = 3).
*p \0.05, statistically different from control. b Survival of C. chanos
versus sub-lethal concentrations of benzo (a) pyrene after 0 h, 14 and
28 days. Values are mean SD (n = 3). *p \0.05, statistically
different from control
Fig. 3 a Feeding rate in C. chanos exposed to sub-lethal concentra-
tions of anthracene. Values are mean SD (n = 3). *p \0.0001,
statistically different from control. b Feeding rate in C. chanos
exposed to sub-lethal concentrations of benzo [a] pyrene. Values are
mean SD (n = 3). *p \0.0001, statistically different from control
Bull Environ Contam Toxicol (2013) 90:6068 63
1 3
anthracene, mean NOEC and LOEC values were 2.03 and
3.09 lg l
-1
respectively and for benzo [a] pyrene, they
were 0.82 and 1.46 lg l
-1
, respectively. Of these two
toxicants, C. chanos was found to be more sensitive to
benzo [a] pyrene (statistically signicant, p \0.05 for both
column and row factor using two way ANOVA) when
compared to anthracene exposure.
Feeding rate in C. chanos exposed to different sub-
lethal concentrations of anthracene and benzo [a] pyrene
is shown in Fig. 3a and b, respectively. Signicant
decrease (p \0.0001) in feeding rate was observed with
increase in both PAHs compounds (data is shown only
for 28 days) in Tables 2 and 3. The feeding rate of
C. chanos exposed to different sub-lethal concentrations
of anthracene and benzo [a] pyrene revealed that the
percentage reduction in feeding rate was statistically
signicant (p \0.0001 for both column and row factor
using two way ANOVA) in sh exposed to benzo
[a] pyrene i.e., 78 % and 74 % after 14 and 28 days of
exposure.
Signicant decrease (p \0.0001) in growth rate was
observed with increasing concentrations of both PAHs
compounds (data is shown only for 28 days in Tables 4 and
5) when compared to control animals. The results of
growth rate of C. chanos exposed to different sub-lethal
concentrations of anthracene and benzo [a] pyrene revealed
that the percentage reduction in growth rate was high
(statistically signicant; p \0.05 for both row and column
factor using two way ANOVA) in sh exposed to benzo
[a] pyrene after 14 and 28 days exposure (Fig. 4a and b).
Percentage reduction in growth rate was 99.7 % and 99.8 %
in the maximum exposed concentration of benzo [a] pyrene
after 14 and 28 days exposure respectively. The results
indicate that benzo [a] pyrene was more toxic than
anthracene to C. chanos in affecting the growth rate
(p \0.05 for both column and row factor using two ways
Table 2 One way analysis of variance in feeding rate for C. chanos
exposed to anthracene after 28 days
(df) F p R
2
6 (between)
14 (within)
20 (total)
10,170 0.0001 0.999
Tukeys multiple comparison tests
Column Mean difference q
Control vs S. control 1.00 3.00 (ns)
Control vs 0.92 60.00 180.00 (s)
Control vs 2.03 62.00 186.00 (s)
Control vs 3.09 68.33 205.00 (s)
Control vs 5.98 74.00 222.00 (s)
Control vs 11.17 77.00 231.00 (s)
S. control vs 0.92 59.00 177.00 (s)
S. control vs 2.03 61.00 183.00 (s)
S. control vs 3.09 67.33 202.00 (s)
S. control vs 5.98 73.00 219.00 (s)
S. control vs 11.17 76.00 228.00 (s)
0.92 vs 2.03 2.00 6.00 (s)
0.92 vs 3.09 8.33 25.00 (s)
0.92 vs 5.98 14.00 42.00 (s)
0.92 vs 11.17 17.00 51.00 (s)
2.03 vs 3.09 6.33 19.00 (s)
2.03 vs 5.98 12.00 36.00 (s)
2.03 vs 11.17 15.00 45.00 (s)
3.09 vs 5.98 5.66 17.00 (s)
3.09 vs 11.17 8.66 26.00 (s)
5.98 vs 11.17 3.00 9.00 (s)
S. control solvent control, ns not signicant, s signicant
Table 3 One way analysis of variance in feeding rate for C. chanos
exposed to benzo [a] pyrene after 28 days
(df) F p R
2
6 (between)
14 (within)
20 (total)
12,870 0.0001 0.999
Tukeys multiple comparison tests
Column Mean difference q
Control vs S. control 0.33 0.93 (ns)
Control vs 0.29 69.33 194.60 (s)
Control vs 0.82 75.00 210.50 (s)
Control vs 1.46 83.67 234.80 (s)
Control vs 2.57 87.00 244.10 (s)
Control vs 4.73 92.67 260.00 (s)
S. control vs 0.29 69.00 193.60 (s)
S. control vs 0.82 74.67 209.50 (s)
S. control vs 1.46 83.33 233.90 (s)
S. control vs 2.57 86.67 243.20 (s)
S. control vs 4.73 92.33 259.10 (s)
0.29 vs 0.82 5.66 15.90 (s)
0.29 vs 1.46 14.33 40.22 (s)
0.29 vs 2.57 17.67 49.58 (s)
0.29 vs 4.73 23.33 65.48 (s)
0.82 vs 1.46 8.66 24.32 (s)
0.82 vs 2.57 12.00 33.67 (s)
0.82 vs 4.73 17.67 49.58 (s)
1.46 vs 2.57 3.33 9.35 (s)
1.46 vs 4.73 9.00 25.26 (s)
2.57 vs 4.73 5.66 15.90 (s)
S. control solvent control, ns not signicant, s signicant
64 Bull Environ Contam Toxicol (2013) 90:6068
1 3
ANOVA). The NOEC and LOEC values were also esti-
mated using growth rate after 28 days. Similar to survival
data, for anthracene, mean NOEC and LOEC values were
2.03 and 3.09 lg l
-1
respectively and for benzo [a] pyrene,
they were 0.82 and 1.46 lg l
-1
, respectively.
Signicant decrease (p \0.0001) in gross conversion
efciency rate was observed with increasing concentrations
of both PAHs compounds (data is shown only for 28 days in
Tables 6 and 7) when compared to control animals. The
gross conversion efciency of C. chanos exposed to differ-
ent concentrations of anthracene and benzo [a] pyrene
showed negative correlation (statistically signicant;
p \0.05 for both column and row factor using two way
ANOVA) with increasing sub-lethal concentrations (Fig. 5a
and b). The percentage reduction in gross conversion ef-
ciencies was 98.5 % and 99.5 % after 14 and 28 days
exposure respectively. In benzo [a] pyrene it was 97.6 %and
99.7 % after 14 and 28 days of exposure respectively.
Discussion
It has been estimated that 10 %80 % of PAH inputs to the
worlds oceans is from atmospheric sources. As a conse-
quence, urban run-off contains PAHs deposited on sur-
faces, as well as mobile-related PAHs from gasoline and oil
drips or spills, exhaust products, tyre particles, and bitumen
from road surfaces (Srogi 2007). Many environments
contain toxic compounds at levels that are too low to induce
an acute lethal response, but may impact the dynamics of
populations by adversely affecting the physiological per-
formance of individual organisms (Muller et al. 2010).
Feeding rate is important for the growth, feed conver-
sion, nutrient retention efciency and chemical composi-
tion of sh (Du et al. 2006). Feeding and feeding
frequencies are key factors that determine the growth and
survival chances of sh. The basic principle in feeding is
that sh should be fed exactly to satiation (FEAP 2010). A
Table 4 One way analysis of variance in growth rate for C. chanos
exposed to anthracene after 28 days
(df) F p R
2
6 (between)
14 (within)
20 (total)
765.8 0.0001 0.997
Tukeys multiple comparison tests
Column Mean difference q
Control vs S. control 1.00 4.35 (ns)
Control vs 0.92 12.00 52.23 (s)
Control vs 2.03 12.83 55.86 (s)
Control vs 3.09 13.75 59.85 (s)
Control vs 5.98 13.84 60.24 (s)
Control vs 11.17 14.61 63.58 (s)
S. control vs 0.92 11.00 47.88 (s)
S. control vs 2.03 11.83 51.51 (s)
S. control vs 3.09 12.75 55.50 (s)
S. control vs 5.98 12.84 55.89 (s)
S. control vs 11.17 13.61 59.23 (s)
0.92 vs 2.03 0.83 3.62 (s)
0.92 vs 3.09 1.75 7.61 (s)
0.92 vs 5.98 1.84 8.00 (s)
0.92 vs 11.17 2.60 11.35 (s)
2.03 vs 3.09 0.91 3.99 (s)
2.03 vs 5.98 1.00 4.38 (s)
2.03 vs 11.17 1.77 7.71 (s)
3.09 vs 5.98 0.09 0.39 (s)
3.09 vs 11.17 0.85 3.72 (s)
5.98 vs 11.17 0.76 3.33 (s)
S. control solvent control, ns not signicant, s signicant
Table 5 One way analysis of variance in growth rate for C. chanos
exposed to benzo [a] pyrene after 28 days
(df) F p R
2
6 (between)
14 (within)
20 (total)
432.6 0.0001 0.994
Tukeys multiple comparison tests
Column Mean difference q
Control vs S. control 1.50 4.74 (ns)
Control vs 0.29 12.60 39.81 (s)
Control vs 0.82 13.53 42.76 (s)
Control vs 1.46 14.60 46.13 (s)
Control vs 2.57 14.80 46.76 (s)
Control vs 4.73 14.98 47.34 (s)
S. control vs 0.29 11.10 35.07 (s)
S. control vs 0.82 12.03 38.02 (s)
S. control vs 1.46 13.10 41.39 (s)
S. control vs 2.57 13.30 42.02 (s)
S. control vs 4.73 13.48 42.60 (s)
0.29 vs 0.82 0.93 2.94 (s)
0.29 vs 1.46 2.00 6.31 (s)
0.29 vs 2.57 2.20 6.95 (s)
0.29 vs 4.73 2.38 7.53 (s)
0.82 vs 1.46 1.06 3.37 (s)
0.82 vs 2.57 1.26 4.00 (s)
0.82 vs 4.73 1.45 4.58 (s)
1.46 vs 2.57 0.20 0.63 (s)
1.46 vs 4.73 0.38 1.21 (s)
2.57 vs 4.73 0.18 0.57 (s)
S. control solvent control, ns not signicant, s signicant
Bull Environ Contam Toxicol (2013) 90:6068 65
1 3
restricted feeding rate will cause impaired health or slow
growth (Du et al. 2006). Small sh used in the present
study have a high energy demand. The present study shows
that growth rate, feeding rate and gross conversion ef-
ciency are being inuenced by sub-lethal concentrations of
anthracene and benzo [a] pyrene. Food intake is a prime
factor inuencing the growth by developing appetite which
is found to be affected by pollutants (Du et al. 2006). It can
be assumed that increasing sub-lethal concentration
decreases the food utilization (Liess et al. 2001). It is well
recognized that toxicants induce many biochemical chan-
ges in the animals preceding cellular and systemic dys-
functions (Surendranath 2003).
Similarly Eertman et al. (1995) reported a signicant
reduction in feeding rate in Mytilus edulis exposed to benzo
[a] pyrene when compared to control mollusk. The present
study indicates the inefcient feeding behavior may be
associated with impairment in absorption, conversion and
metabolism and suggests that reduction in feeding rate i.e.,
food intake, may be due to loss of apetite and/or damage to
taste receptors. Similar ndings have also been obtained by
Jee et al. (2004) in olive ounder, Paralichthys olivaceus
when exposed to sub-lethal concentrations of phenanthrene.
The growth rate of C. chanos was found to be negatively
correlated with increasing sub-lethal concentrations of
anthracene and benzo [a] pyrene. The reduction in growth
rate was 99.7 % and 99.8 % for anthracene and benzo
[a] pyrene respectively after 28 days of exposure. Similarly
Kim et al. (2008) estimated the sub-lethal effect of dietary
benzo [a] pyrene on growth changes in juvenile rocksh,
Sebastes schlegeli under running water test system (ow
rate1.2 l min
-1
) after 30 days. The inhibition of gross
conversion efciency of C. chanos exposed to sub-lethal
concentrations of both PAHs may be due to the lowered
assimilation efciency which effectively reduced the food
for maintenance and growth (Ford 2005).
Fig. 4 a Growth rate of C. chanos exposed to sub-lethal concentra-
tions of anthracene. Values are mean SD (n = 3). *p \0.0001,
statistically different from control. b Growth rate of C. chanos
exposed to sub-lethal concentrations of benzo [a] pyrene. Values are
mean SD (n = 3). *p \0.0001, statistically different from control
Table 6 One way analysis of variance in gross conversion efcieny
for C. chanos exposed to anthracene after 28 days
(df) F p R
2
6 (between)
14 (within)
20 (total)
334.7 0.0001 0.993
Tukeys multiple comparison tests
Column Mean difference q
Control vs S. control 0.60 2.26 (ns)
Control vs 0.92 7.60 28.72 (s)
Control vs 2.03 9.00 34.02 (s)
Control vs 3.09 10.20 38.55 (s)
Control vs 5.98 11.88 39.69 (s)
Control vs 11.17 7.00 44.92 (s)
S. control vs 0.92 8.40 26.46 (s)
S. control vs 2.03 9.60 31.75 (s)
S. control vs 3.09 9.90 36.28 (s)
S. control vs 5.98 11.28 37.42 (s)
S. control vs 11.17 1.40 42.65 (s)
0.92 vs 2.03 2.60 5.29 (s)
0.92 vs 3.09 2.90 9.82 (s)
0.92 vs 5.98 4.28 10.96 (s)
0.92 vs 11.17 1.20 16.19 (s)
2.03 vs 3.09 1.50 4.53 (s)
2.03 vs 5.98 2.88 5.66 (s)
2.03 vs 11.17 0.30 10.90 (s)
3.09 vs 5.98 1.68 1.13 (s)
3.09 vs 11.17 1.38 6.36 (s)
5.98 vs 11.17 0.76 5.22 (s)
S. control solvent control, ns not signicant, s signicant
66 Bull Environ Contam Toxicol (2013) 90:6068
1 3
The induction of physiological modications by sub-
lethal doses provides more realistic and more varied
information on the actual toxicity of pollutants (Gaudy
et al. 1991; Manyin and Rowe 2009). Such physiological
modications disturb the energetic equilibrium of organ-
isms (Gaudy et al. 1991) and thus can have long term
effects on the stability of these populations. The imbalance
can arise from decreased nutritional energy from food
intake because of decreased assimilation capacities, or
from increased respiratory requirements as a result of
stress. Of these two toxicants, C. chanos was found to be
more sensitive to benzo [a] pyrene when compared to
anthracene exposure. Limited studies have been carried out
in anthracene and benzo [a] pyrene for the determination of
safe level estimates such as NOEC and LOEC. Similarly,
Hall and Oris, (1991) has reported the NOEC and LOEC
values for anthracene were 6 and 12 lg l
-1
.
Present study is the rst attempt to determine the safe
limits and effects of PAHs viz., anthracene and benzo
[a] pyrene in milksh C. chanos. These ndings deserve
further attention as they highlight the important role of
individual biomarkers of susceptibility on PAHs induced
biochemical changes in target organs.
Acknowledgments One of us L. Palanikumar, kindly acknowledges
the receipt of fellowship grant from Ministry of Earth Sciences
ICMAM, Chennai, India. Authors sincerely thank Dr. R. Babu
Rajendran, Associate Professor, Department of Environmental Bio-
technology, Bharathidasan University, Trichy, India for providing
GCMS facility.
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