evaluation of polychaetes, perinereis cultrifera (grube, 1840)

Transcript

Speciation and flow of pollutants
EVALUATION OF POLYCHAETES,
PERINEREIS CULTRIFERA (GRUBE, 1840), AS
INDICATORS OF SEDIMENT MICRO-ORGANIC
CONTAMINATION
Nicoletta Nesto, Daniele Cassin, Luisa Da Ros
Istituto di Scienze Marine, CNR, Venezia
Riassunto
Dati preliminari sul possibile utilizzo del polichete Perinereis cultrifera, specie
diffusa in laguna di Venezia, come indicatore della contaminazione da
inquinanti organici nei sedimenti, sono stati ottenuti da esperimenti di
laboratorio. A tal fine, esemplari adulti di policheti provenienti da un sito di
riferimento, sono stati sperimentalmente esposti per 14 e 25 giorni a due
sedimenti naturali diversamente contaminati da PCB ed IPA. La risposta
biologica è stata valutata in termini di variazioni percentuali della
concentrazione corporea di ciascun inquinante e di malondialdeide, un
metabolita cellulare che segnala stress ossidativo.
I risultati hanno indicato in generale un significativo bioaccumulo di tutti i
congeneri di PCB nei policheti esposti per 25 giorni al sedimento più
contaminato, diversamente da quelli esposti al sedimento di controllo, in cui è
stato evidenziato un trend alla diminuzione. Dopo 14 giorni di esposizione ad
entrambi i sedimenti gli organismi hanno invece mostrato trend discordanti. Le
variazioni percentuali del contenuto di IPA sono risultate molto differenti, in
relazione ai singoli composti esaminati, ma in generale è stata evidenziata una
chiara tendenza alla diminuzione, spiegabile parzialmente con la elevata
capacità detossificante degli organismi per questi composti.
La percentuale di variazione del contenuto di Malondialdeide è risultata
maggiore nei campioni esposti per 25 giorni ad entrambi i sedimenti, non
essendo stata evidenziata alcuna differenza tra i campioni esposti ai due tipi di
sedimenti. Questo risultato indica che tale parametro può essere influenzato da
molteplici fattori ambientali, e va quindi considerato un indicatore di stress
generico.
Questi risultati, pur preliminari, indicano che il polichete P. cultrifera può essere
considerato un potenziale indicatore della presenza di PCB nei sedimenti
lagunari.
Abstract
This study aims to evaluate the possible use of the polychaetes, Perinereis
cultrifera, as indicators of organic contamination in the sediments of the Lagoon
of Venice. Organisms from a reference site were exposed in laboratory to
169
Scientific Research and Safeguarding of Venice 2007
natural sediments differently contaminated by PCBs and PAHs for 14 and 25
days. The biological responses were evaluated as percentage variations
relating to controls of the body burden of each contaminant and of
malondialdehyde (MDA), a metabolite indicating oxidative stress within the
organisms.
The results showed a significant bioaccumulation of each PCB congener in
worms exposed for 25 days to the most contaminated sediment, whereas a
slight depletion was evidenced in worms exposed to the reference sediment. On
the contrary, the samples exposed for 14 days to both sediments evidenced
more erratic trends. The PAH concentrations resulted highly variable according
to each different compound; however, their reduction was more repeatedly
observed, following the induction of the efficient enzymatic systems able to
metabolised them. The results of MDA content demonstrated that also in this
species this metabolite should be considered as a generic stress index,
influenced by various environmental and endogenous disturbing factors.
Although preliminary, these data indicate that the polychaete P. cultrifera may
be a promising indicator of the sediment PCB contamination in the Venice
Lagoon.
1
Introduction
The sediments of the Lagoon of Venice may be considered a sink, source and
cycling centre for micro-organic pollutants, therefore the assessment of their
contamination level holds an important role in the management of the Lagoon
ecosystem, although it does not necessarily reflect the bioavailable fraction of
these compounds. For this reason, the evaluation of the pollutant content in the
biota, which reflects different types of exposure route, physiological status and
age of the organism, is an integrated parameter and therefore may be a more
useful tool in defining the quality status of an environmental compartment.
Among a great variety of biomonitor organisms which have been proposed and
used in the last decades in biomonitoring surveys in the framework of both
national and international research programmes, the polychaetes represent a
large group of bottom dwelling organisms able to accumulate in their tissues
organic xenobiotics from water and sediment (Magnusson et al., 2006;
Cornellissen et al., 2006; Ruus et al., 2005; Maruya et al., 1997; Meador et al.,
1997; Means & McElroy, 1997; Driskoll & McElroy, 1996). Moreover, these
organisms are important preys of several bottom dwelling fish species, and
therefore may contribute to the transfer of contaminants to higher levels in the
food chain (Ruus et al., 2002; 2005). The polychaetes belonging to Neredidae
family, for their wide spatial distribution, food web position, and a relatively long
life cycle, generally characterized by a singular reproduction event, were
included as bioaccumulation indicators in US-EPA official procedures (US-EPA,
1995). Due to the lack of organic pollutant bioaccumulation data in the
polychaetes of the Venice Lagoon, the aim of this study was to verify the
possible use as organic pollutant biomonitor, to date relatively unexplored, of
the Neredidae polychaetes Perinereis cultrifera. This species is widely
170
distributed within the lagoon, and therefore it might be a more suitable sentinel
organism than the most studied Hediste diversicolor, which is found
predominantly along the inner borders of the lagoon. P. cultrifera is a
gonochoric species characterized by semelparous reproductive strategy which
is preceded by epitokous metamorphosis, and it lives in sandy silt sediments at
a maximum depth of 15 cm. In Lagoon of Venice these worms can reach 11 cm
length and reproduce in March (Prevedelli & Simonini, 2003).
In the framework of the Corila Research Program 2004-2006 - Research Line
3.8. Speciation, distribution, fluxes, bioaccumulation and toxicity of the main
contaminants in the Lagoon of Venice, a laboratory experiment was performed
to assess the bioaccumulation ability of P. cultrifera for organic pollutants.
Moreover, to evaluate the well-being of the organisms, the malondialdehyde
content (MDA), was determinate as a generic biomarker of oxidative stress
(Gérard-Monnier et al., 1998).
2
2.1
Materials and Methods
Sediment collection and preparation
A long-term sediment laboratory assay was performed in May 2006 using
sediments collected from two differently impacted areas of the Lagoon: Tresse
(T) as a polluted site and Palude della Rosa (PR) as a reference. Composite
subtidal surface sediment samples (5-10 cm) were collected from each site
using a Van Vleet grab, transferred to the laboratory and then sieved (1.5 mm
mesh size) to remove indigenous animals and large debris. The sediments were
stored at 4°C in darkness for two weeks before the initiation of the assay. Four
plastic aquaria (30x20x13 cm) were prepared for testing each sediment type. A
3 cm layer of sieved sediment was placed at the bottom of each aquarium and
afterwards 3 litres of filtered seawater (36 PSU) were carefully added. After a
conditioning period of 2 hours, aeration was provided using plastic tips
suspended 2 cm above the sediment surface. Subsamples of the two field
sediments were stored at -20°C until chemical analyses.
2.2
Polychaetes collection and preparation
About 300 organisms (5±1 cm length) were collected at low tide at a pristine site
in the Lagoon. Once in laboratory, they were rinsed in sea water and put in
aerated acquaria containing quartz sand and sea water at 20°C, 34±1 PSU for a
two-weeks acclimatization period, during which the organisms were deprived of
food and subjected to a photoperiod of 12h light and 12 h dark.
2.3
Sediment exposure design
Thirty acclimated individuals were carefully added to each test aquarium. They
were kept to a constant temperature of 20°C, with a photoperiod 12 h light:12 h
dark, and no food was supplied during the assay. For each sediment trial, a
subsample of 50-60 individuals were randomly recovered at different time: T0
(reference sample after the acclimatization period), T14 (after 14 days
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exposure) and T25 (after 25 days exposure) and differently processed
according to the various chemical and biological analyses.
2.4
Chemical and biological analyses
Polyaromatic hydrocarbons (PAH) and chlorinated hydrocarbons (PCBs) were
Soxhlet-extracted from both organisms (after a 3 days depuration period in
aquaria containing quartz sand and sea water) and sediments for eight hours
with n-hexane. The extract was evaporated at 50°C to constant weight for the
determination of Extractable Organic Matter (EOM) and, after dissolution in 1
cm3 n-hexane, fractionated by chromatography on an alumina/silica gel column.
PCBs from thee first fractions of column eluates were analysed by ECD gas
chromatography (C. Erba 4160 GC) using a 30 m x 0.32 mm i.d. SE-54 fused
silica column with hydrogen as a carrier gas. The concentration of 14 USEPA
priority pollutant PAHs were analyzed with high performance liquid
chromatograph (HP 1090, USA) on a reverse-phase column (Supelcosil LCPAH 250 mm x 2.1 mm 5 μm) with a programmed fluorescence detector.
The content of MDA was determined spettrophotometrically in 3 pools of 0.5 g
of minced frozen worms according to the method described by Gèrard-Monnier
et al. (1998). The evaluation of MDA is widely used as indicative of lipid
peroxidation and it is based on the rapid reaction between the 1-Methyl-2phenylindole with MDA yelding a stable carbocyanine dye with maximal
absorption wavelength of 586 nm. MDA content was estimated using the
tetramethoxypropane as reference standard.
The results of both chemical and biological analyses were expressed, for each
group of exposed organisms, as percentage variations with regard to the control
(at time 0)
3
Results
The test sediments used in the exposure experiment resulted differently
contaminated by PCBs and PAHs (Tab.1). The PCBs content in the sediment
from Tresse (T) was approximately 10-fold higher than the sediment from
Palude della Rosa (PR). PCB 153, PCB 138 resulted the most abundant
congeners at both sites representing roughly the 30% and 20 % of the total
PCBs. The PAHs contamination in sediment from Tresse was 35-fold higher
than the reference one (Palude della Rosa), showing that pyrene and
fluoranthene were the most abundant compounds at both sites. In T sediment
they represented the 25% and 19% respectively, of total PAHs and in PR
sediment the 19% and 14%.
The percentage variations of PCBs (individual PCB congeners, Sum and
Arochlor 1254+1260) in exposed polychaetes are presented in Fig. 1. In
general, the most remarkable results were recorded after 25 days, when a
marked bioaccumulation for all the examined PCB congeners (except PCB 52)
and Arochlor 1254+1260 was identified in polychaetes exposed to T sediment,
whereas a small reduction was evidenced in the sample exposed to PR
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sediment. After 14 day exposure results showed less and erratic variations. The
PAH percentage variations resulted different in relation to each individual PAH,
although a general reduction was observed in samples exposed to both
sediments (Fig. 2). In particular, phenanthrene and benzo(a)anthracene
resulted slightly bioaccumulated only in sample exposed to PR sediment for 14
days, fluoranthene only in sample exposed to PR sediment for 25 days, and
benzo(b)fluoranthene only in sample exposed to T sediment for 14 days.
Anthracene was marked bioaccumulated in samples exposed to both sediments
only for 14 days, whereas pyrene resulted differently bioaccumulated in the
samples exposed to T sediment, showing higher bioaccumulation level in
organisms exposed for 14 days. All the other PAHs were lower than in the
reference organisms for both sediments.
The percentage variation of MDA resulted higher in samples exposed to both
sediments for 25 days, accounting for an increase of 29% and 23% in Tresse
and Palude della Rosa sediments, respectively (Fig. 3). After 14 day exposure
the variations in MDA content were lower and exhibited opposite trends in the
two sediments.
Microorganic
pollutants
T
PR
PCB 52
2.48 ± 1.22
0
PCB 101
3.15 ± 1.38
0.30 ± 0.03
PCB 110
1.95 ± 0.17
0.46 ± 0.01
PCB 118
3.08 ± 0.47
0.29 ± 0.03
PCB 153
10.30 ± 2.04
0.96 ± 0.27
PCB 138
6.38 ± 2.11
0.50 ± 0.09
PCB 180
5.02 ± 3.09
0.29 ± 0.14
Sum PCB
32.37 ± 3.96
2.81 ± 0.28
Ar 1254+1260
67.16 ± 3.90
6.30 ± 1.05
Naphthalene
0.00
0.00
Acenaphthylene
0.00
0.00
Acenaphthene
5.57 ± 0.23
0.00
Fluorine
30.88 ± 4.33
0.00
338.85 ± 44.27
5.85 ± 1.41
18.68 ± 5.04
2.20 ± 0.02
Phenanthrene
Anthracene
Fluoranthene
673.41 ± 40.25
14.93 ± 2.75
Pyrene
889.69 ± 377.95
18.47 ± 10.67
Benz[a]anthracene
202.21 ± 16.59
9.25 ± 1.18
Chrysene
223.97 ± 41.46
9.96 ± 1.84
Benzo[b]fluoranthene
283.24 ± 38.36
13.16 ± 3.45
Benzo[k]-fluoranthene
203.52 ± 11.95
8.71 ± 1.00
Benzo[a]pyrene
314.82 ± 33.83
9.24 ± 2.88
28.20 ± 4.14
0.64 ± 0.08
357.60 ± 82.62
6.68 ± 2.12
diBenzo[a,h]anthracene
Benzo-[g,h,i]perylene
Indeno[1,2,3-cd]pyrene
Sum PAHs
0.00
0
3570.64 ±
620.24
99.08 ± 25.71
Tab 1 – Organic
micropollutants content (ng
g-1 dw) in sediments from
Tresse (T) and Palude della
Rosa (PR), used in the
exposure experiment.
173
400
%
T T14
350
PR T14
T T25
PR T25
300
250
200
150
100
50
0
A
r1
25
4+
1
26
PC
B
0
-150
Su
m
PC
B
13
B
PC
B
PC
18
8
3
15
8
PC
PC
PC
B
B
11
11
1
B
B
10
52
-50
-100
0
0
PC
Fig 1 – PCB content in
samples of P. cultrifera
exposed for 14 and 25 days
to different contaminated
sediments (T= Tresse, PR=
Palude della Rosa). Values
are expressed as percentage
variations with regard to the
time 0 control.
Legend: T T14= sample exposed to Tresse sediment for 14 days; PR T14= sample
exposed to Palude della Rosa sediment for 14 days; T T25= sample exposed to Tresse
sediment for 25 days; PR T25= sample exposed to Palude della Rosa sediment for 25
days.
80
%
T T14
60
PR T14
T T25
PR T25
40
20
Phe
Ant
0
B[a]A
Ft
Py
B[b]Ft
Chy
B[a]Py
B[k]Ft
B[ghi]Per
diB[a,h]A
SumPAHs
-20
Fig 2 – PAH content in
time 0 control.
-40
-60
-80
-100
days.
PAHs abbreviation: Phe= phenanthrene; Ant= anthracene; Ft= fluoranthene; Py =pyrene;
B[a]A= benz[a]anthracene; Chy= chrysene; B[b]Ft= benzo[b]fluoranthene; B[k]Ft=
174
benzo[k]-fluoranthene; B[a]Py= benzo[a]pyrene; diB[a,h]A= dibenz[a,h]anthracene;
B[g,h,i]Per= benzo-[g,h,i]perylene.
40
%
MDA
30
20
10
Fig 3 – MDA content in
time 0 control.
0
-10
T T14
PR T14
T T25
PR T25
-20
-30
days.
4
Discussion
The long-term exposure to different contaminated sediments highlighted that P.
cultrifera is able both to bioaccumulate PCBs and to metabolize the PAHs. In
particular, all the examined PCB congeners resulted actively bioaccumulated
after 25 day in the most contaminated sediment, apart from the less chlorinated
compound, PCB 52, possibly because more dissolved in water than associated
with particles, due to its low octanol/water partition coefficient (Log Kow =5.84)
(Ruus et al., 2002). On the contrary, the organisms exposed to the reference
sediment for 25 days exhibited a marked reduction of all PCB congeners. The
hexa- and hepta-chlorinated biphelyls, i.e. PCB 153, PCB138 and PCB 180,
which are characterized by a moderate hydrophobicity, resulted the most
bioaccumulated, and were in agreement with previous studies dealing with
infaunal organisms (Goerke & Weber, 1990; Meador et al., 1997; Pruell et al.,
2000; Ruus et al., 2005). As these type of congeners are the most abundant in
both test sediments, it may be suggested that the accumulation is mainly
related to the ingested sediments, although the direct absorption through the
cuticle could not been excluded (Fowler et al., 1978). The low and erratic
variations of the PCB congeners after 14 day exposure to both sediments
suggests that the steady-state for the tissue residues may be reached after a
longer exposure time, as already observed for Hediste diversicolor, for which
the EPA procedures recommend a 28 days test (US-EPA, 1995).
The variations of PAHs contents in P. cultrifera resulted quite different for each
considered compound, but a clear reduction was generally evidenced in both
sediments, due to their low environmental persistence and the rapid induction of
175
specific detoxifing enzymatic system (Christensen et al., 2002; Driscoll &
McElroy, 1996; Forbes et al., 1996; McElroy, 1990).
The PAH compounds, accumulated indifferently after 14 or 25 days from both
sediments, were phenanthrene, anthrancene, fluoranthene, pyrene and
benz(a)anthracene. Previous studies indicated that they are highly
bioaccumulated in different polychaetes specie as Arenicola marina,
Abarenicola pacifica, Nereis diversicolor and highlighted a wide variability of
bioaccumulation factors among various species, in relation to different feeding
and behavioural strategies and/or routes of exposure (Cornelissen et al., 2006;
Christensen et al., 2002; Weston, 1990; Augenfeld & Anderson, 1982). It has
also been suggested that compounds with log Kow around 5 have the highest
bioaccumulation factors in comparison with contaminants with lower or higher
log Kow as the former are quickly eliminated and the last are not bioavailable
due to extensive sorption to particulate matter in the sediments (Christensen et
al., 2002).
The higher MDA content recorded in samples exposed for 25 days to both
sediments, indicated that also in polychaetes, like in other invertebrate taxa, this
metabolite may be considered as a generic stress biomarker, influenced by
numerous confounding factors. A very marked intraspecific agonistic behaviour,
reported for this species by Scaps (1995) might have been an important
stressing factor for the individuals maintained in each aquarium.
Conclusions
The long term exposure experiment showed a significant bioaccumulation of all
PCB congeners in worms exposed for 25 days to the most contaminated
sediment, whereas a slight depletion was evidenced in worms exposed to the
reference sediment. According to these preliminary results, P. cultrifera might
be a promising suitable bioindicator of the PCBs in the lagoon sediments. The
PAH concentrations resulted highly variable in relation to the different
compounds, although in general a depletion was observed in worms exposed to
both sediments, possibly because they are metabolized by efficient enzymatic
systems.
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evaluation of polychaetes, perinereis cultrifera (grube, 1840)

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