Ecological Indicators 73 (2017) 698–707

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Ecological Indicators
journal homepage: www.elsevier.com/locate/ecolind

Urbanization effects on different biological organization levels of an

estuarine polychaete tolerant to pollution
Wilson Antonio Weis a,∗ , Carlos Henrique Lemes Soares b ,
Daiane Paula Cunha de Quadros c , Mauana Scheneider c , Paulo Roberto Pagliosa a,d
a
Universidade Federal de Santa Catarina, Núcleo de Estudos do Mar, 88040-900 Florianópolis, SC, Brazil
b
Universidade Federal de Santa Catarina, Departamento de Bioquímica, 88040-900 Florianópolis, SC, Brazil
c
Universidade Federal de Santa Catarina, Departamento de Química, 88040-900 Florianópolis, SC, Brazil
d
Universidade Federal de Santa Catarina, Departamento de Geociências, Laboratório de Biodiversidade e Conservação Marinha, 88040-900 Florianópolis,
SC, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Estuarine species exposed to diffuse contaminants might trigger either positive or negative feedbacks in
Received 4 July 2016 many biological scales. Their life history traits performing at different biological organization levels could
Received in revised form 25 October 2016 propose an organism as a useful indicator of environmental pollution, mainly addressed as sensitive or
Accepted 26 October 2016
tolerant species. To track the effects of contaminants from the molecular to the population level of the
Available online 6 November 2016
polychaete Laeonereis acuta we utilize a framework of biomarkers. For this purpose we assessed the L.
acuta frequency of micronuclei at the molecular level, the body size and biomass at individual level, and
Keywords:
the production-to-biomass ratio at population level in five urbanized and five non-urbanized estuaries in
Bioindicator
Biomarker
southern Brazil. L. acuta had significantly varying positive and negative feedbacks between urbanized and
Body size non-urbanized estuaries at multiple biological scales. These generalized effects in all biological organiza-
Estuarine pollution tion scales indicate a pollution impact on the polychaete. The main responses accounted for individuals
Micronuclei becoming lengthy and weighty, but with molecular damage. The L. acuta allocation of energy to body
Turnover ratio enlargement in polluted environments, and a consequent reduced population turnover, contradicts the
expected from an opportunist species. The damages in DNA and the internal strategies of individuals, as
antioxidant defense mechanisms, could favor resistance of the population and tolerance to pollutants.
All of these characteristics induce bioaccumulation and could cause bottom-up pollution transfer com-
promising the estuarine food web. These results, ascertain that L. acuta could be considered as a tolerant
species, instead of an opportunistic, and as a useful indicator of environmental pollution in estuaries.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction life traits with adaptive values (fitness) that are nowadays cou-
pled with or benefited from contaminants (Kawecki and Ebert,
Coastal urbanization has increased the discharge of diffuse toxic 2004). Notwithstanding, species exposed to contaminants usually
substances into water bodies responsible for acute and chronic present negative responses in genetic, physiological, morphologi-
degradation of the estuarine biota. In advanced situations, this cal and behavioral levels (Mersch et al., 1996; Fleeger et al., 2003;
process causes elimination of sensitive species and dominance of Schiedek et al., 2006; Catalano et al., 2012). The description of bio-
the most tolerant or opportunistic individuals (Klerks and Weis, logical mechanisms under such evolutionary and ecological scales
1986; Bickham et al., 2000; D’Alessandro et al., 2015). Along has been a current challenge to elucidate how species function as
their evolutionary history, tolerant species might have selected tolerants to environmental pollution.
Evaluation of the effects of contaminants usually involves an
outlined bulk of biomarkers (Monserrat et al., 2007). To integrally
comprehend those effects, the biomarkers need to be processed in
∗ Corresponding author.
many biological scales, as species may experience stress at differ-
E-mail addresses: [email protected], wilson [email protected]
(W.A. Weis), [email protected] (C.H.L. Soares),
ent levels of biological organization throughout the medley from
[email protected] (D.P.C. de Quadros), molecular to the community level. Among the implications to the
[email protected] (M. Scheneider), [email protected] environmental management of investigating many biological orga-
(P.R. Pagliosa).

http://dx.doi.org/10.1016/j.ecolind.2016.10.029
1470-160X/© 2016 Elsevier Ltd. All rights reserved.
 W.A. Weis et al. / Ecological Indicators 73 (2017) 698–707 699

nization levels, the assessment of changes in lower biological levels

can be used to avoid further effects on higher levels, when they have
not yet happened. However, the uses and responses of biomarkers
in molecular or subcellular levels in invertebrates are still poorly
E10
known and fragmented (Dixon et al., 2002). Conversely, the expres-
sion of both qualitative and quantitative damage to DNA is very
similar between invertebrates and higher complexity organisms
(Dixon et al., 2002; Hagger et al., 2002; Jha, 2004). These similar-
E4
ities could be used to drive endeavors in researches with marine
invertebrates.
Polychaetes numerically dominate the estuarine communities
living in the water-sediment interface, have a short lifespan and
are less motile than other benthic macroinvertebrates, and thus
responsive to changes in their surrounding environment. All of
these characteristics confer the generic status of good indicators
of environmental health (Durou et al., 2007; Botter-Carvalho et al.,
2011). The Nereididae polychaete Laeonereis acuta is widely spread
in South American Atlantic estuaries and sheltered areas, from
2◦ S (Brazil) to 42◦ S (Argentina) (Orensanz and Gianuca, 1974;
Pamplin et al., 2007). Early studies have indicated L. acuta to have
an opportunistic behavior because of their relatively short life
cycle and rapid sediment recolonization strategies (Netto and Lana,
1994; Omena and Amaral, 2000). Further studies highlighted the
species as pertaining to a tolerant assemblage in urbanized estu- E9
aries (Pagliosa and Barbosa, 2006). This was suggested after their
E2
density and biomass were found to be related to the general state E1
of the environment, both in water and sediments, than simply to E3
E5
modifications in the sediment. More recently, the potentiality of L.
E7
acuta as a tolerant bioindicator of pollution has stimulated the map- E8 E6
ping of their histological and morphological alterations, as well as
biochemical responses, accumulation, and biotransformation after
exposure to contaminants (Geracitano et al., 2002; Ferreira-Cravo
et al., 2008; Ventura-Lima et al., 2011). However, an integrated
biomarkers assessment at different levels of biological organization
Fig. 1. Map indicating the 10 estuaries in Southern Brazil. E1-Costeira do Pirajubaé,
of L. acuta under urbanization effects is still lacking. E2–Itacorubi River, E3-Maruim River, E4-Saguaçu Bay, E5-Aririu River, E6-Ribeirão
The aim of this study was to assess the effects of diffuse pol- da Ilha, E7-Lagoinha do Leste, E8-Massiambu River, E9-Ratones River, E10-Sai Mirim
lution caused by urbanization in estuaries at different biological River.
organization levels of the polychaete L. acuta. For this purpose we
used the biomarkers frequency of micronuclei at subcellular level,
body size and biomass at the individual level, and production-to-
biomass ratio at the population level. This framework was applied recorded: 12 ␮M of P, 217 ␮M of N, 46 mg/kg of Cu, 56 mg/kg of
in urbanized and non-urbanized estuaries. Thus, we expected L. Pb, 144 mg/kg of Zn) (Pagliosa, 2005; Pagliosa et al., 2006a,b, 2016;
acuta biomarkers to indicate the environmental health differences Rovai et al., 2013). Then, we performed a visual interpretation of
between estuaries types which would be detected in all levels aerial images complemented by field surveys, and used existent
of biological organization. It is hypothesized that L. acuta from data of water and sediment quality to select. In this sense, we have
urbanized estuaries will exhibit higher frequencies of micronu- determined two types of estuaries for our study in relation to the
clei in cells, lower individual body sizes and biomass, as well as degree of anthropogenic environmental disturbance: (i) urbanized
higher production-to-biomass ratio in populations when compared estuaries, whenever available, indicated by the water and sediment
to those from non-urbanized estuaries. data, and when a great extent of the estuarine basins was composed
of residential, commercial or industrial areas with clear signs of
disturbed landscape; and (ii) non-urbanized estuaries, whenever
2. Material and methods available, indicated by the water and sediment data, and when the
estuarine basin presents just a small or absence of signs of anthropic
The study was conducted from August to September in 2014 interventions and visually the physiognomy of the landscape is
at ten estuaries located between the coordinates 25◦ 5 S–48◦ 3 W preserved. The study estuaries were spaced from 05 km to 200 km
and 27◦ 5 S–48◦ 4 W (Fig. 1). The coastal region is typically com- and belonged to a different watershed, avoiding confound effect of
posed of bights founded by quaternary sediments turning over urbanization.
marine, tidal, and river-lake plains crossed by estuaries. The cli- In order to assess the responses of L. acuta to environmental con-
mate is humid subtropical with rainfall well distributed throughout ditions, three sites were randomly established in shallow waters at
the year. The annual mean temperature is 20 ◦ C with seasonal dif- the mouth of five urbanized and five non-urbanized estuaries. The
ferences ranging from 17 to 22 ◦ C. The tidal regime is microtidal mouths of the estuaries were chosen for sampling because they sink
with average amplitude of 0.83 m for spring tides and 0.15 m for the continental material and might therefore represent the general
neap tides (Cruz, 1998). The estuarine basins in south Brazil are health of these small systems. At each site, individuals of L. acuta,
generally well preserved areas (highest concentrations recorded: sediment and water were sampled. The effects of urbanization on
1 ␮M of P, 52 ␮M of N, 27 mg/kg of Cu, 28 mg/kg of Pb, 105 mg/kg different levels of biological organization of the polychaete were
of Zn) interspersed with urbanized areas (highest concentrations assessed using: (i) the frequency of micronuclei, at the molecular
700 W.A. Weis et al. / Ecological Indicators 73 (2017) 698–707

level; (ii) the body size and biomass, at the individual level; and (iii) individuals in a population instead of sampling individuals of the
the production-to-biomass ratio (P/B), at the population level. population living in a specified area), we used previously published
information on L. acuta population (Omena and Amaral, 2000). The
2.1. Molecular level data set is made of two year-long monthly samples and with L.
acuta populations structured in four different cohorts. We esti-
At each site, multiple individuals of L. acuta were carefully and mated the P/B ratios based on mean individual biomass (mg) and
manually collected using a spatula and tweezers, and held in a based on mean individual biomass per square meter (mg/m2 ). The
container with sediment and water from the local source. In the comparison between the P/B ratios calculated with sample-based
laboratory, all containers were maintained with aeration. Then, we data and individual-based data were found to be not significant
randomly chose 10 alive and unharmed organisms (no breakage (ANOVA, F(1,40) = 0.080; P = 0.778). We therefore assumed that a
during handling) from each sampling site to evaluate the frequency production-to-biomass ratio using individual-based data could be
of micronuclei in cells. The posterior segments of alive individ- used to represent population level test in our study.
uals were removed using a scalpel and were kept for four days
in aquarium (natural variability of light and temperature) receiv-
2.4. Environmental data
ing dehydrated brine shrimp (Branchipus stagnalis) as food. At the
end of this period, the regenerated tissues were removed, dis-
At each estuary and site, samples of near-bottom water were
aggregated into acetic acid and methanol at a ratio of 3:1 and
collected in 500 mL polyethylene bottle, previously cleaned by
centrifuged at 900 rpm for four minutes. The bulk of the mate-
immersion in 50% HCl, washed with deionized water and kept
rial resulting from the disaggregation of the 10 specimens was
refrigerated at 4 ◦ C. Determination of total nitrogen, total phos-
fixed in methanol and stained with May-Grünwald Giemsa 5% and
phorus and total phenols was performed by spectrophotometry
diluted in phosphate buffer for 10 min. Micronuclei frequencies
according to Standard Methods (APHA, 1998), using a UV visi-
were evaluated under a light microscope (Olympus CX21SF1, 100×
ble (FEMTO spectrophotometer 700 Plus). For the particle size
magnification) by counting one thousand cells per site (bulk of
analyses, we used homogenized sediment sub-samples of a stan-
10 individuals). Micronuclei were considered as the corpuscles of
dardized weight (50 g). The sand fraction was assessed by dry
smaller diameters, entirely separated and which were similar in
sieving using meshes among −1.5 and 4.0 Phy, and fine fractions
color and morphology to the main nuclei. The micronuclei test was
were accessed via pipetting at 20 ◦ C (Suguio, 1973). The aluminum,
adapted from Fenech (2000).
cadmium, chromium, copper and lead contents in sediments were
obtained by contrAA 600 high resolution continuum source atomic
2.2. Individual level absorption spectrometer (HR-CS GF-AAS, Analytik Jena AG, Jena,
Germany) with a transversely heated graphite tube atomizer. The
From the container, we randomly select 10 other alive and samples digestions were preceded with addition of 2.5 mL HNO3
unharmed organisms from each sampling site to measure for indi- 67% and 7.5 mL HCl 37% for approximately 3.0 g, using a TOPwave
vidual size and biomass. The body length was taken from the IV laboratory microwave digestion system (Analytik Jena AG, Jena,
anterior prostomium to posterior pygidium of each individual Germany). The standard solution of Al, Cd, Cr, Cu and Pb were
under a Zeiss optical stereoscope (Discovery V12; 10 ␮m precision) prepared from stock concentrate solution containing 1000 mg L−1
coupled to a digital camera (AxioCam MRc 5). Additionally, the wet (Merck, Darmstadt, Germany) with nitric acid were used to pre-
weight of each individual was measured (BEL Analytical balance; pare the external calibration curve. Additionally, to provide a basis
0.0001 g precision and internal calibration M124AI 24 V 550 mA). for asserting the absence of sediment toxicity (as expected for non-
urbanized estuaries) we contrasted the metal concentrations found
2.3. Population level with the Consensus-Based Threshold Effect Concentration (TEC;
MacDonald et al., 2000). Similarly, to asserting sediment toxic-
The biomass values of sampled individuals from each site were ity (as expected for urbanized estuaries) we contrasted the metal
used to calculate empirically the production-to-biomass ratio of concentrations found with the Consensus-Based Probable Effect
the population. We used the following formula adapted from the Concentration (PEC; MacDonald et al., 2000).
model of Brey (2012):

log10 P/B = 7.947(−2.294 log10 M − 2409.856 × (1/(T + 273)) 2.5. Data analysis
+ 0.548 + 582851 × (log10 M × (1/(T + 273)))
In order to assess the relationship between samples of the
biomarkers at multiple biological scales of the polychaete and
Where, P = production, B = mean population biomass, M = mean samples of environmental conditions of estuaries, a distance-
individual biomass (we used a global data bank on body composi- based redundancy analysis (dbRDA; Legendre and Anderson, 1999;
tion of aquatic organisms to get the energy content (Joule) per mg McArdle and Anderson, 2001) was used. In this analysis, the axes
of wet mass; Brey et al., 2010), and T = mean seasonal temperature of ordinations are linear combinations of the environmental vari-
(we used the historical, from 1960 to 2015, mean temperature value ables that best explain the biological variation. The Bray-Curtis
of 17.6 ◦ C from the sampled months for all sites; INMET (2016). The coefficient and Euclidean distance were used on squared-root
value 0.548 is a product of the adapted model, since all organisms transformed biological and environmental data, respectively. To
belong to Annelida (taxon = 1), are infaunal (mobility = 1), and occur model the changes in each of the biological variables represent-
in intertidal habitats (depth = 0 m). ing different biological organization levels (micronuclei frequency,
The population P/B ratio is the renovation or replacement of body size, biomass, and P/B ratio) related to the environmental con-
biological material, thus being the quantity of matter and energy ditions, we used a canonical analysis of principal coordinates (CAP;
potentially available for higher trophic level. The empiric model Anderson and Wills, 2003). The CAP analyses how well the mul-
of Brey (2012) used to calculate population productivity was built tivariate environmental data can predict the position of samples
using sample-based data and was originally treated as a scale of along a continuous values of each biomarker. CAP avoids over-
area (mg/m2 ). In order to validate the performance of the empiri- parameterization (i.e., to avoid including too many axes and finding
cal model using individual-based data (i.e., sampling a number of spurious relationships) by choosing the number of principal coor-
 W.A. Weis et al. / Ecological Indicators 73 (2017) 698–707 701

Urbanized Estuaries
Non-urbanized Estuaries

dbRDA (11.3% of fitted, 10% of tottal variation)

dbRDA (88.8% of fitted, 77.2 of total variation)

Fig. 2. Distance-based RDA ordination relating sediment and water parameters to L. acuta responses (micronuclei frequency, body size, biomass, and P/B ratio) in urbanized
and non-urbanized estuaries.

dinate axes that minimize a leave-one-out residual sum of squares urbanized samples appeared to group with content of copper and
(Anderson and Robinson, 2003). very poorly sorted grains (large variance).
Additionally, the biological and environmental variables were All recorded metal concentrations in non-urbanized estuaries
individually evaluated using hierarchical nested analyses of vari- were lower than the Consensus-Based Probable Effect Concen-
ance. The factor “condition” was fixed (two levels: urbanized vs tration (PEC) and just one estuary (estuary 6) reported higher
non-urbanized) and the factor “estuary” was random and nested concentrations than the Consensus-Based Threshold Effect Concen-
within the condition factor (five levels: five estuaries within each tration (TEC), indicating an absence of sediment toxicity (Fig. 3).
estuary type). The three sites within each estuary provided the Conversely, the chromium, copper, and lead concentrations in all
replicates. The nested design provides an estimate of the con- sites of urbanized estuaries (except estuary 5 for copper) were
tribution of each estuary to the total variation among replicates higher than the TEC and some estuaries (mainly for chromium)
within the urbanized and non-urbanized comparison. Partitioning were higher than the PEC, indicating the presence of sediment tox-
of the variances associated with each factor permits unconfounded icity.
comparisons at any of the chosen factors (Underwood, 1997). Addi- The analysis of variances of environmental variables showed
tionally, components of variation were calculated to estimate the significantly higher values for aluminum, cadmium, lead and
percentage of variability attributed to each factor and for the sorting in sediment as well as nitrogen in water from urban-
residual by restricted maximum likelihood estimation (Pinheiro ized estuaries than from non-urbanized ones (Tables 1 and 2;
and Bates, 1996). Data were previously tested for heterogeneity Figs. 3 and 4). The magnitude of the effects for all of those variables
of variance with the Cochran test and squared-root transformed presented the greatest percentage of explanation related to the
whenever necessary. For the ANOVAs, we used the GAD pack- factor condition (all components of variation >50%). Neither envi-
age (Sandrini-Neto and Camargo, 2012) in the R environment (R ronmental variable showed higher values in non-urbanized than
Development Core Team, 2013), and for the dbRDA and CAP we urbanized estuaries and the percentage of sand, content of cop-
used the PERMANOVA+ add-on PRIMER software (Anderson and per and chrome in sediment, and concentration of phosphorus and
Gorley, 2007). phenols in water were not different between the two estuary con-
ditions. The lowest values of components of variation (between 27
and 42%) highlighted the low importance of factor conditions for
3. Results these variables. Contrarily, all variables from sediment and water
were significantly different between estuaries nested within con-
The spatial distribution of L. acuta samples was distinct between dition. This means that the variation within a group of estuaries is
the urbanized and non-urbanized estuaries, while three replicates different in general to the variation within the other group of estu-
from a non-urbanized estuary appeared in an intermediate posi- aries. In general, the urbanized estuaries were more heterogeneous
tion (dbRDA with 89.6% of the explained variation; Fig. 2). Samples among themselves than non-urbanized estuaries (mean and error
from urbanized estuaries were characterized by higher content of in Figs. 3 and 4). The magnitude of the effects of the factor estuary
metals (lead, cadmium, and chrome) in sediment and higher con- nested in factor condition was low when the factor condition for the
centrations of dissolved nutrients (phosphorus and nitrogen) and same variable was significant (between 23 and 33%) and increased
phenol in water. Otherwise, biological samples of non-urbanized only a little when the factor condition was not significant (between
estuaries were more related with sandy sediments and well sorted 34 and 51%), indicating the low importance of the variation within
grains (low variance). Nevertheless, the intermediate group of non-
702 W.A. Weis et al. / Ecological Indicators 73 (2017) 698–707

. . . . . . . . . . . . . . TEC Table 1
_ _ _ _ _ _ _ _ _ PEC Result of hierarchical nested analysis of variance and component of variation (CV)
of metal from sediments in urbanized and non-urbanized (Condition) estuaries.
80

Source Anova CV (%)

60
Cr (µg/g)

Df MS F-ratio P-value

Al
40

Condition 1 0.071 15.418 0.004 57.038

Estuary(Condition) 8 0.005 34.818 <0.001 33.103
20

Residuals 20 <0.001 9.859

Cd
0

Condition 1 0.138 12.686 0.007 50.059

Estuary(Condition) 8 0.011 8.806 <0.001 30.829
Residuals 20 0.001 19.112
0.030

Cr
Condition 1 36.744 3.624 0.093 37.578
Estuary(Condition) 8 10.140 67.556 <0.001 51.490
Al (µg/g)
0.020

Residuals 20 0.150 10.932

Cu
Condition 1 12.989 4.536 0.065 41.701
0.010

Estuary(Condition) 8 2.864 91.362 <0.001 49.314

Residuals 20 0.031 8.985
0.000

Pb
Condition 1 9.280 13.329 0.006 52.626
Estuary(Condition) 8 0.696 14.977 <0.001 32.375
Residuals 20 0.047 14.999
0.12

In bold P < 0.05, Df = degrees of freedom, MS = mean square.

0.08
Cd (µg/g)

Table 2
Result of hierarchical nested analysis of variance and component of variation (CV)
of sediment and water parameters in urbanized and non-urbanized (Condition)
0.04

estuaries.

Source Anova CV (%)

0.00

Df MS F-ratio P-value

SEDIMENT
Sand
20

Condition 1 62.368 4.337 0.070 38.151

Estuary(Condition) 8 14.380 25.297 <0.001 45.767
Residuals 20 0.568 16.082
15
Cu (µg/g)

Grain Sorting
Condition 1 3.765 12.778 0.007 49.717
10

Estuary(Condition) 8 0.295 7.851 <0.001 30.260

Residuals 20 0.037 20.023
5

WATER
N
0

Condition 1 0.020 16.980 0.003 49.880

Estuary(Condition) 8 0.001 3.211 0.016 23.153
Residuals 20 <0.001 26.967
14

P
12

Condition 1 0.088 3.356 0.104 27.796

10

Estuary(Condition) 8 0.026 3.358 0.013 33.929

Pb (µg/g)

Residuals 20 0.008 38.275

8

Phenol
6

Condition 1 0.010 2.473 0.155 26.587

4

Estuary(Condition) 8 0.004 9.900 <0.001 46.446

Residuals 20 <0.001 26.966
2

In bold P < 0.05, Df = degrees of freedom, MS = mean square.

0

E1 E2 E3 E4 E5 E6 E7 E8 E9 E10

Urbanized Non−urbanized
conditions urbanized and non-urbanized, and among the factor
Fig. 3. Metal sediment toxicity (mean ± 1SD) in urbanized and non-urbanized estu- estuaries nested in conditions (Table 3; Fig. 5). The lower levels
aries. Underlines represent the Consensus-Based Threshold Effect Concentration of biological organization (frequency of micronuclei at molecular
(TEC) and the Consensus-Based Probable Effect Concentration (PEC), according to level; body size and biomass at individual level) showed higher
MacDonald et al. (2000). See Fig. 1 for original names of estuaries.
values in urbanized than in non-urbanized estuaries. Contrarily, the
higher level of biological organization assessed, P/B ratio of popu-
groups of estuaries to explain all variability found in the analysis of lation, was significantly higher in non-urbanized estuaries than in
variance. urbanized ones. The magnitudes of the effects of the factor con-
All parameters presenting the different levels of biological dition were always high (between 54 and 66%) and of the factor
organization of L. acuta were significantly different between the estuary were always low (between 15 and 33%). These values of
 W.A. Weis et al. / Ecological Indicators 73 (2017) 698–707 703

Table 3
Result of hierarchical nested analysis of variance and component of variation (CV) of
L. acuta different levels of biological organization in urbanized and non-urbanized
(Condition) estuaries.

Source Anova CV (%)

Df MS F-ratio P-value

Micronuclei frequency (1000 cells−1 )

Condition 1 11.981 13.837 0.005 54.195
Estuary(Condition) 8 0.866 20.948 <0.001 33.006
Residuals 20 0.041 12.800

Body size (cm)

Condition 1 1.778 35.095 <0.001 59.456
Estuary(Condition) 8 0.051 3.387 0.013 19.114
Residuals 20 0.015 21.430

Biomass (mg)
Condition 1 7.783 66.136 <0.001 66.537
Estuary(Condition) 8 0.118 3.013 0.021 15.069
Residuals 20 0.039 18.394

P/B ratio (y−1 )

Condition 1 17.115 26.764 <0.001 60.415
Estuary(Condition) 8 0.639 10.422 0.013 25.306
Residuals 20 0.061 14.279

In bold P < 0.05, Df = degrees of freedom, MS = mean square.

ties representative of the urbanized and non-urbanized estuaries

(Fig. 6). The squared canonical correlation was high and positive for
micronuclei frequency (␦2 = 0.86), body size (␦2 = 0.79) and biomass
(␦2 = 0.93), and high and negative for the P/B ratio (␦2 = 0.82). Addi-
tionally, the CAP analysis for micronuclei frequency showed three
replicated sites of the non-urbanized estuaries were intermediately
grouped among sites of the urbanized estuaries.

4. Discussion

L. acuta had significantly varying positive and negative feed-

backs between urbanized and non-urbanized estuaries at multiple
biological scales. These results indicate that the biomarkers showed
the differences in environmental health of the studied estuaries
in southern Brazil quite well. In fact, the environmental condi-
tions of urbanized estuaries were exceeding the consensus-based
threshold effect concentration (TEC) and in some sites even sur-
passed the consensus-based probable effect concentration (PEC).
Conversely, non-urbanized estuaries never reached the PEC levels
and rarely surpassed the TEC levels. Then, we confirm our hypoth-
esis that biomarkers can detect environmental health differences
between estuaries types in all levels of biological organization stud-
ied. The detected generalized effects in all biological organization
scales indicate a pollution impact on L. acuta. The population of
the pollution tolerant polychaete is growing more, the individu-
als are increasing in body mass and body size, but the molecules
are standing damaged. Furthermost, there is a comprehensive field
investigation stressing changes in estuarine soft-bottom commu-
nities connected with changes in L. acuta population, expressed as
increased abundance, size and biomass (Souza et al., 2016; Pagliosa
and Barbosa, 2006; Souza et al., 2013; Brauko et al., 2015; Gusmão
et al., 2016; Magalhães and Barros, 2011). On the other hand, there
Fig. 4. Sediment and water variables (mean ± 1SD) in urbanized and non-urbanized
is a collection of laboratory experiments evidencing changes in L.
estuaries. See Fig. 1 for original names of estuaries. acuta behavior, histology (coelom obliteration, separation of the
cuticle from epidermis), tissue (loss of the digestive epithelium)
and biochemical parameters (catalase, superoxide dismutase, lipid
component of variation indicate that contribution of the variation peroxidation, and glutathione S-transferase) caused by different
within groups of estuaries is less important for the total spatial pollutants (Geracitano et al., 2002; Ferreira-Cravo et al., 2008; Leão
variability found than the variation between the urbanized and et al., 2008; Ventura-Lima et al., 2011). All of these evidences evoke
non-urbanized condition. two questions about L. acuta responses to pollution: (i) at the higher
The CAP analyses found a strong linear relationship among each scales of biological organization (from individual to community),
L. acuta biological parameters and the sediment and water proper- the polychaete has been efficiently occupying niche space which is
704 W.A. Weis et al. / Ecological Indicators 73 (2017) 698–707

Urbanized Estuaries
15
Non-urbanized Estuaries

10

0
-0.4 -0.2 0 0.2 0.4

1
-0.4 -0.2 0 0.2 0.4

800

600

400

200

0
-0.4 -0.2 0 0.2 0.4

9.5

8.5

7.5

6.5

5.5
-0.4 -0.2 0 0.2 0.4

Fig. 6. Canonical analysis of principal coordinates (CAP) to model the relationships

Fig. 5. Biomarkers of L. acuta different levels of biological organization (mean ±1SD)
of sediment and water variables to L. acuta responses (micronuclei frequency, body
in urbanized and non-urbanized estuaries. See Fig. 1 for original names of estuaries.
size, biomass, and P/B ratio) in urbanized and non-urbanized estuaries.

vague for other species that are more sensitive to pollution. The ∼36% more than others nereidids) and the consequent increased
advantage comes mostly from changing phenotypic expressions potential for chromosomal mutations related to inversions (4-fold
related with body size and biomass. As found in other invertebrates more nucleoli per interphase cell as in other nereidids) (Ipucha
(Hoffmann et al., 2004), these changes in size and biomass could et al., 2007). In fact, L. acuta is so different to other family species
be rooted in the increased L. acuta chromosome numbers (2n = 38; in many aspects that it stands in a separate and unresolved clade
 W.A. Weis et al. / Ecological Indicators 73 (2017) 698–707 705

in phylogenetic analysis of the nereidids (Santos et al., 2006); (ii) species, when usually populations high density and biomass in pol-
at more basal levels of biological organization (from molecule to luted sites are supported by individuals with smaller size ranges
organ) the species developed strategies to deal with excessive pol- and higher production values with a turnover ratio higher than in
lutants and toxins. The substances are accumulated in regions of non-polluted sites or when pollution was ended (Alla et al., 2006;
the body, tissues and cells, and mucus appears to be a key sub- Méndez et al., 1997). All presented evidences in molecular, individ-
stance for primary protection, favoring the biotransformation of ual and populations ascertain L. acuta as a tolerant species, instead
contaminants (Leão et al., 2008). The resultant permanence and of an opportunist, and is a useful indicator of environmental pollu-
resistance of the enlarged organism with accumulated contami- tion in estuaries.
nants in their body are eye-catching prey to consumers (Ieno et al., Our results showed an environmental pollution impact in all
2000), which might cause a bottom-up pollution transfer (biomag- levels of biological organization of L. acuta in urbanized estuar-
nification) along the estuarine food web. ies, which indicate that even in non-heavy polluted estuaries, the
The response mechanisms of organisms to pollution are usu- synergisms of diffuse contaminants could cause a generalized bio-
ally rapidly and evidently observed at molecular and cellular scales logical effect. The main response in higher biological organization
but become rather difficult to define at higher levels due to evolu- levels of the polychaete (above individual level) appears to be
tionary distance and environmental complexity (Smolders et al., changes in their phenotype expression related with increased body
2004; Sulmon et al., 2015). Here, we detected organisms with size and biomass, and the consequent decreased P/B ratio. However,
micronucleus frequency, a type of DNA damage, three times higher what appears to be a competitive advantage at higher biological lev-
in urbanized than in non-urbanized estuaries, indicating general els is high cost at lower levels (bellow individual level), with DNA
genotoxicity of metals in the former (Sanchez-Galan et al., 1999; damage. We detected the effects of contaminants on molecular lev-
Ayllón et al., 2000; Andrade et al., 2004). Further, we recorded els occur earlier, which might favor decision making by managers
L. acuta DNA damage but no individual or population changes in and stakeholder as preventive practices before the emergence of
the sites of one estuary typified as non-urbanized and presenting damages to populations or communities levels. All these life his-
non-expected great concentrations of lead, copper, chromium and tory characteristics of L. acuta point to a tolerance to pollution
aluminum in sediments, indicating an early stage of genotoxicity. performance, which is a desirable feature for a useful bioindicator.
Metals exert genotoxic action alone or through synergistic action. The approach used here to track the effects of contaminants at
In sub-lethal concentrations and chronic form, they could cause multiple biological scales using a framework of biomarkers showed
marked changes in antioxidant enzymes, suggesting the induc- accurate when contrasted with the environmental data. All used
tion of oxidative stress with implications for cell repair systems biomarkers are friendly and easy to use. The micronuclei frequency
(Livingstone, 2003; Nusetti et al., 2005) which affects the DNA is a lower cost and more time efficient to use than other tradi-
molecule (Meneghini, 1997). Among all types of DNA damages, tional molecular approaches, as proteomic ou metabolomic. The
strand break and the subsequent micronucleus formation is the biomarkers at individual level are widespread used and do not
most challenging to the cells once the continuous formation of such need any additional material than the already used in benthic ecol-
breaks may contribute to genomic instability (Halazonetis et al., ogy. The empirical population P/B ratio is a powerful tool already
2008). implemented, but unfortunately scarcely used. All used biomarkers
Estuarine species living in urbanized areas are usually char- seem to be particularly effective to highlight differences in envi-
acterized by small organisms with low individual biomass and ronmental health and could be an alternative method to determine
r-strategists (Elliott and Quintino, 2007). L. acuta is a typical r- estuarine pollution condition.
strategist species with no parental care, and the presence of
females with large mature oocytes and recruits throughout the year Acknowledgments
(Omena and Amaral, 2000; Santos et al., 2006; Martin and Bastida,
2006). Conversely, in the present study, the size and biomass of The authors wish to acknowledge support from the Brazilian
L. acuta individuals at urbanized estuaries were two- to three-fold National Research Council (CNPq). WAW was supported by grants
higher than those of the non-urbanized. Possibly, the exposure of from the CAPES Foundation, Ministry of Education of Brazil.
organisms to sublethal amounts of xenobiotics triggers adaptive
responses providing greater ability to tolerance (Klerks and Weis, References
1987; Adams, 2005). Polychaetes advantages of an increased size
could come from the concentration of metals in the tissues tending APHA, A., 1998. Standard methods for the examination of water and wastewater,
to a decrease in the increased sized and age of organisms (Mendez 20.
Adams, S.M., 2005. Assessing cause and effect of multiple stressors on marine
and Páez-Osuna, 1998), meaning that larger organisms are more systems. Mar. Pollut. Bull. 51, 649–657, http://dx.doi.org/10.1016/j.marpolbul.
likely to successfully thrive under contamination. When subjected 2004.11.040.
to low to moderate concentrations of xenobiotics, L. acuta responds Alla, A.A., Gillet, P., Deutsch, B., Moukrim, A., Bergayou, H., 2006. Response of Nereis
diversicolor (Polychaeta, Nereidae) populations to reduced wastewater
by positively activating detoxifying enzymes in sufficient quan- discharge in the polluted estuary of Oued Souss, Bay of Agadir, Morocco. Estuar.
tities to prevent damage caused by oxidative stress (Geracitano Coast. Shelfı́s 70, 633–642, http://dx.doi.org/10.1016/j.ecss.2006.01.028/t.
et al., 2004a,b). Additionally, the production of mucus substantially Anderson, M.J., Gorley, R.N., 2007. PERMANOVA+ for PRIMER: Guide to Software
and Statistic Al Methods. PRIMER-E, Plymouth, UK.
increases the antioxidant defense system (Moraes et al., 2006). Anderson, M.J., Robinson, J., 2003. Generalized discriminant analysis based on
Thus, living in polluted environment requires many metabolic distances. Aust. N. Z. J. Stat. 45, 301–318, http://dx.doi.org/10.1111/1467-842X.
responses that demands high-energy expenditure to organisms 00285.
Anderson, M.J., Wills, T.J., 2003. Canonical analysis of principal coordinates: a
(Bayne et al., 1979; Smolders et al., 2004). In addition, the poly-
useful method of constrained ordination for ecology. Ecology 84, 511–525,
chaete may take advantage strategy for surviving in areas with an http://dx.doi.org/10.1890/0012-9658(2003)084[0511:CAOPCA]2.0.CO;2.
increased organic material in sediments. Hence, for L. acuta pop- Andrade, V.M., Silva, J., Silva, F.R., Heuser, V.D., Dias, F.J., Yoneama, M.L., Freitas,
T.R.O., 2004. Fish as bioindicators to assess the effects of pollution in two
ulation this excessive energy might be required to increase body
Souther Brazilian rivers using the comet assay and micronucleus test. Environ.
size and biomass coupled with activating responses related, espe- Mol. Mutagen. 44, 459–468, http://dx.doi.org/10.1002/em.20070.
cially, to the antioxidant defense mechanisms. As a consequence of Ayllón, F., Suciu, R., Gephard, S., Juanes, F., Garcia-Vasque, z., 2000. Conventional
energy allocation to tolerate the environmental pollution in urban- armament wastes induce micronuclei in wild brown trout Salmo trutta. Mutat.
Res. 470, 169–176, http://dx.doi.org/10.1016/s1383-5718(00)00101-7.
ized estuaries, the production to biomass ratio of L. acuta tended Bayne, B.L., Moore, M.N., Widdows, J., Livingstone, D.R., Salkeld, P., 1979.
to decrease. That is the contrary to the expected to an opportunist Measurament of the response of individuals to environmental stress and
706 W.A. Weis et al. / Ecological Indicators 73 (2017) 698–707

pollution: studies with bivalve mollusk. Philos. Trans. R. Soc. Lond. B 286, Kawecki, T.J., Ebert, D., 2004. Conceptual issues in local adaptation. Ecol. Lett.,
563–581, http://dx.doi.org/10.1098/rstb.1979.0046. 1225–1241, http://dx.doi.org/10.1111/j.1461-0248.2004.00684.x.
Bickham, J.W., Sandhu, S., Hebert, P.D.N., Chikhi, L., Athwal, R., 2000. Effects of Klerks, K.P., Weis, J.S., 1987. Genetic adaptation to heavy metals in aquatic
chemical contaminants on genetic diversity in natural populations: organism: a review. Environ. Pollut. 45, 173–205, http://dx.doi.org/10.1016/
implications for biomonitoring and ecotoxicology. Mutat. Res. 463, 33–51, 0269-7491(87)90057-1.
http://dx.doi.org/10.1016/S1383-5742(00)00004-1. Leão, J.C., Geracitano, L.A., Monserrat, J.M., Amado, L.L., Yunes, J.S., 2008.
Botter-Carvalho, M.L., Carvalho, P.V.V., Santos, P.J.P., 2011. Recovery of Microcystin-induced oxidative stress in Laeonereis acuta (Polychaeta
macrobenthos in defaunated tropical estuarine sediments. Mar. Pollut. Bull. 62, Nereididae). Mar. Environ. Res. 66, 92–94, http://dx.doi.org/10.1016/j.
1867–1876, http://dx.doi.org/10.1016/j.marpolbul.2011.04.044. marenvres.2008.02.033.
Brauko, K.M., Souza, F.M., Muniz, P., Camargo, M.G., Lana, P.C., 2015. Spatial Legendre, P., Anderson, M.J., 1999. Distance-based redundancy analysis: testing
variability of three benthic indices for marine quality assessment in a multispecies responses in multifactorial ecological experiments. Ecol. Monogr.
subtropical estuary of Southern Brazil. Mar. Pollut. Bull. 91, 454–460. 69, 1–24, http://dx.doi.org/10.1890/0012-9615(1999)069[0001:DBRATM]2.0.
Brey, T., Müller-Wiegmann, C., Zittier, Z.M.C., Hagen, W., 2010. Body composition CO;2.
in aquatic organisms—a global data bank of relationships between mass, Livingstone, D.R., 2003. Oxidative stress in aquatic organisms in relation to
element composition., and energy content. J. Sea Res. 64, 334–340. pollution and aquaculture. Rev. Med. Vet-Toulouse 154, 427–430.
Brey, T., 2012. A multi-parameter artificial neural network model to estimate MacDonald, D.D., Ingersoll, C.G., Berger, T.A., 2000. Development and evaluation of
macrobenthic invertebrate productivity and production. Limnol. Oceanogr. consensus-based sediment quality guidelines for freshwater ecosystems. Arch.
Methods 10, 581–589, http://dx.doi.org/10.4319/lom.2012.10.581. Environ. Contam. Toxicol. 39, 20–31, http://dx.doi.org/10.1007/
Catalano, B., Moltedo, G., Martuccio, G., Gastaldi, L., Virno-Lamberti, C., Lauria, A., s002440010075.
Ausili, A., 2012. Can Hediste diversicolor (Nereidae, Polychaete) be considered Magalhães, W.F., Barros, F., 2011. Structural and functional approaches to describe
a good candidate in evaluating PAH contamination? A multimarker approach. polychaete assemblages: ecological implications for estuarine ecosystems.
Chemosphere 86, 875–882, http://dx.doi.org/10.1016/j.chemosphere.2011.10. Mar. Freshwa. Res. 62, 918–926, http://dx.doi.org/10.1071/MF10277.
040. Martin, J.P., Bastida, R., 2006. Population structure, growth and production of
Cruz, O., 1998. A Ilha de Santa Catarina e o continente próximo; um estudo de Laeonereis culveri (Nereididae: Polychaeta) in tidal flats of Rio de la Plata
geomorfologia costeira. Editora da UFSC, Florianópolis, Brasil, 276p. estuary, Argentina. J. Mar. Biol. Assoc. U.K. 86, 235–244, http://dx.doi.org/10.
D’Alessandro, L.A., Hoehme, S., Henney, A., Drasdo, D., Klingmüller, U., 2015. 1017/S0025315406013087.
Unraveling liver complexity from molecular to organ level: challenges and McArdle, B.H., Anderson, M., 2001. Fitting multivariate models to community data:
perspectives. Prog. Biophys. Mol. Biol. 117, 78–786, http://dx.doi.org/10.1016/j. a comment on distance-based redundancy analysis. Ecology 82, 290–297,
pbiomolbio.2014.11.005. http://dx.doi.org/10.1890/0012-9658(2001)082[0290:FMMTCD]2.0.CO;2.
Dixon, D.R., Pruski, A.M., Dixon, L.R., Jha, A.N., 2002. Marine invertebrate Mendez, N., Páez-Osuna, F., 1998. Trace metals in two populations of the fireworm
eco-genotoxicology: a methodological overview. Mutagenesis 17, 495–507. Eurythoe complanata from Mazatlán Bay: effect of body size on concentrations.
Durou, C., Poirier, L., Amiard, J.C., Budzinski, H., Gnassia-Barelli, M., Lemenach, K., Environ. Pollut. 102, 279–285, http://dx.doi.org/10.1016/S0269-
Peluhet, L., Mouneyrac, C., Roméo, M., Amiard-Triquet, C., 2007. Biomonitoring 7491(98)00061-X.
in a clean and a multi-contaminated estuary based on biomarkers and Meneghini, R., 1997. Iron homeostasis, oxidative stress, and DNA damage. Free
chemical analyses in the endobenthic worm Nereis diversicolor. Environ. Pollut. Radic. Biol. Med. 23, 783–792, http://dx.doi.org/10.1016/S0891-
148, 445–458, http://dx.doi.org/10.1016/j.envpol.2006.12.022. 5849(97)00016-6.
Elliott, M., Quintino, V., 2007. The estuarine quality paradox, environmental Mersch, J., Beauvais, M.N., Nagel, P., 1996. Induction of micronuclei in haemocytes
homeostasis and the difficulty of detecting anthropogenic stress in naturally and gill cells of zebra mussels Dreissena polymorpha, exposed to clastogens.
stressed areas. Mar. Pollut. Bull. 54, 640–645. Mutat. Res. 371, 47–55, http://dx.doi.org/10.1016/S0165-1218(96)90093-2.
Fenech, M., 2000. The in vitro micronucleus technique. Mutat. Res. 455, 81–95, Monserrat, J.M., Martínez, P.E., Geracitano, L.A., Amado, L.L., Martins, C.M.G., Pinho,
http://dx.doi.org/10.1016/S0027-5107(00)00065-8. G.L.L., Chaves, I.S., Soares, I., Ferreira-Cravo, M., Ventura-Lima, J., Bianchini, A.,
Ferreira-Cravo, M., Ventura-Lima, J., Sandrini, J.Z., Amado, L.L., Geracitano, L.A., 2007. Pollution biomarkers in estuarine animals: critical review and new
Rebelo, M., Bianchini, A., Monserrat, J.M., 2008. Antioxidant responses in perspectives. Comp. Biochem. Phys. C 146, 221–234, http://dx.doi.org/10.1016/
different body regions of the polychaeta Laeonereis acuta (Nereididae) exposed j.cbpc.2006.08.012.
to copper. Ecotoxicol. Environ. Saf. 72, 388–393, http://dx.doi.org/10.1016/j. Moraes, B.M., Ferreira, J.L.R., Ros, a.C.E., Sandrini, J.Z., Votto, A.P., Trindade, G.S.,
ecoenv.2008.07.003. Geracitano, L.A., Abreu, P.C., Monserrat, J.M., 2006. Antioxidant properties of
Fleeger, J.W., Carman, K.R., Nisbet, R.M., 2003. Indirect effects of contaminants in the mucus secreted by Laeonereis acuta (Polychaeta, Nereididae): A defense
aquatic ecosystems. Sci. Total Environ. 317, 207–233, http://dx.doi.org/10. against environmental pro-oxidants? Comp. Biochem. Phys. C 142, 293–300,
1016/S0048-9697(03)00141-4. http://dx.doi.org/10.1016/j.cbpc.2005.10.017.
Geracitano, L., Monserrat, J.M., Bianchini, A., 2002. Physiological and antioxidant Netto, S.A., Lana, P.C., 1994. Effects of sediment disturbance on the structure of
enzyme responses to acute and chronic exposure of Laeonereis acuta benthic fauna in a subtropical tidal creek of southeastern Brazil. Mar. Ecol.
(Polychaeta, Nereididae) to copper. J. Exp. Mar. Biol. Ecol. 277, 145–156, http:// Prog. Ser. 106, 239–247.
dx.doi.org/10.1016/S0022-0981(02)00306-4. Nusetti, O., Zapata-Vivenes, E., Escalpés, M.M., Rojas, A., 2005. Antioxidant
Geracitano, L., Monserrat, J.M., Bianchini, A., 2004a. Histological and morphological Enzymes and tissue regeneration in Eurythoe complanata (Polychaeta:
alterations induced by copper exposure in Laeonereis acuta (Polychaeta, Amphinomidae) exposed to used vehicle crankcase oil. Arch. Environ. Contam.
Nereididae). Mar. Environ. Res. 58, 263–267, http://dx.doi.org/10.1016/j. Toxicol. 48, 509–514, http://dx.doi.org/10.1007/s00244-004-0041-0.
marenvres.2004.03.069. Omena, E.P., Amaral, A.C.Z., 2000. Population dynamics and secondary production
Geracitano, L., Monserrat, J.M., Bianchini, A., 2004b. Oxidative stress in Laeonereis of Laeonereis acuta (Treadwell, 1923) (Nereididae: Polychaeta). Bull. Mar. Sci.
acuta (Polychaeta Nereididae): environmental and seasonal effects. Mar. 67, 421–431.
Environ. Res. 58, 625–630, http://dx.doi.org/10.1016/j.marenvres.2004.03.053. Orensanz, J.M., Gianuca, N.M., 1974. Contribuição ao conhecimmento dos anelídeos
Gusmão, J.B., Brauko, K.M., Eriksson, B.K., Lana, P.C., 2016. Functional diversity of poliquetas do Rio Grande do Sul, Brasil, 1: Lista sistemática preliminar e
macrobenthic assemblages decreases in response to sewage discharges. Ecol. descrição de três novas espécies. Rev. Mus. Argent. Cienc. Nat. 4, 1–37.
Indic. 66, 65–75. Pagliosa, P.R., Barbosa, F.A.R., 2006. Assessing the environment-benthic fauna
Hagger, J.A., Fisher, A.S., Hill, S.J., Depledge, M.H., Jha, A.N., 2002. Genotoxic, coupling in protected and urban areas of southern Brazil. Bio. Conserv. 129,
cytotoxic and ontogenetic effects of tri-n-butyltin on the marine worm, 408–417, http://dx.doi.org/10.1016/j.biocon.2005.11.008.
Platynereis dumerilii (Polychaeta: Nereidae). Aquat. Toxicol. 57, 243–255, Pagliosa, P.R., Fonseca, A., Barbosa, F.A.R., 2006a. Evidence of systemic changes in
http://dx.doi.org/10.1016/S0166-445X(01)00200-4. trace metal concentrations in subtropical estuarine sediments as a result of
Halazonetis, T.D., Gorgoulis, V.G., Bartek, J., 2008. An oncogene-induced DNA urbanization. J. Coast. Res. SI39, 1078–1083.
damage model for cancer development. Science 319, 1352–1355, http://dx.doi. Pagliosa, P.R., Fonseca, A., Barbosa, F.A.R., Braga, E., 2006b. Urbanization impact on
org/10.1126/science.1140735. subtropical estuaries: a comparative study of water properties in urban areas
Hoffmann, A.A., Sgrò, C.M., Weeks, A.R., 2004. Chromosomal inversion and in protected areas. J. Coast. Res. SI39, 731–735.
polymorphisms and adaptation. Trends Ecol. Evol. 19, 482–488, http://dx.doi. Pagliosa, P.R., Oortamn, M.S., Rovai, A.S., Soriano-Sierra, E.J., 2016. Is mangrove
org/10.1016/j.tree.2004.06.013. planting insufficient for benthic macrofaunal recoverywhen environmental
Instituto Nacional de Metrologia (INMET). Banco de Dados Meteorológicos para stress is persistent? Ecol. Eng. 95, 290–301, http://dx.doi.org/10.1016/j.
Ensino e Pesquisa. (Disponível em: ecoleng.2016.06.036.
http://www.inmet.gov.br/portal/index.php?r=bdmep/bdmep). (accessed Pagliosa, P.R., 2005. Another diet of worms: the applicability of polychaete feeding
18.09.16). guilds as a useful conceptual framework and biological variable. Mar. Ecol. 26,
Ieno, E., Martin, J.P., Bastida, R., 2000. Estiamtion of size classes in Laeonereis acuta 246–254, http://dx.doi.org/10.1111/j.1439-0485.2005.00065.x.
(Polychaeta: Nereididae) based on jaw length and body width usable in trophic Pamplin, P.A.Z., Almeida, T.C.M., Silva-Filho, J.P., 2007. New record of Laeonereis
studies. Bull. Mar. Sci. 67, 39–43. acuta (Treadwell, 1923) (Nereididae: Polychaeta) in Northeast coast of Brazil.
Ipucha, M.C., Santos, C.G., Lana, P.C., Sbalqueiro, I.J., 2007. Cytogenetic Biota Neotrop. 7, 353–355, http://dx.doi.org/10.1590/S1676-
characterization of seven South American species of nereididae (Annelida: 06032007000300035.
Polychaeta): implications for the karyotypic evolution. J. Basic Appl. Genet. 18, Pinheiro, J.C., Bates, D.M., 1996. Unconstrained parametrizations for
27–38. variance-covariance matrices. Stat. Comput. 6, 289–296, http://dx.doi.org/10.
Jha, A.N., 2004. Genotoxicological studies in aquatic organisms: an overview. 1007/BF00140873.
Mutat. Res. 552, 1–17, http://dx.doi.org/10.1016/j.mrfmmm.2004.06.034.
 W.A. Weis et al. / Ecological Indicators 73 (2017) 698–707 707

R Development Core Team, 2013. R: A Language and Environment for Statistical Souza, F.M., Brauko, K.M., Lana, P.C., Muniz, P., Camargo, M.G., 2013. The effect of
Computing. R Foundation for Statistical Computing, Vienna, Austria http:// urban sewage on benthic macrofauna: a multiple spatial scale approach. Mar.
www.R-project.org. Pollut. Bull. 67, 234–240, http://dx.doi.org/10.1016/j.marpolbul.2012.10.021.
Rovai, S.A., Barufi, J.B., Pagliosa, P.R., Scherner, F., Torres, M.A., Horta, P.A., Souza, F.M., Brauko, K.M., Gilbert, E.R., Martins, C.C., Lana, P.C., Camargo, M.G., 2016.
Simonassi, J.C., Quadros, D.P.C., Borges, D.L.G., Soriano-Sierra, E.J., 2013. Complex spatial and temporal variation of subtropical sewage impact. Mar.
Photosynthetic performance of restored and natural mangroves under Environ. Res. 116, 61–70, http://dx.doi.org/10.1016/j.marenvres.2016.02.008.
different environmental constraints. Environ. Pollut. 181, 233–241, http://dx. Suguio, K., 1973. Introdução à sedimentologia. Editora Edgar Blucher Ltda. São
doi.org/10.1016/j.envpol.2013.06.023. Paulo-SP. 317.
Sanchez-Galan, S., Linde, A.R., Garcia-Vasquez, E., 1999. Brown trout and European Sulmon, C., Baaren, J.V., Cabello-Hurtado, F., Gouesbet, G., Hennion, F., Mony, C.,
Minnow as target species for genotoxicity tests: differential sensitivity to Renault, D., Bormans, M., El Amrani, A., Wiegand, C., Gérard, C., 2015. Abiotic
heavy metals. Ecotoxicol. Environ. Saf. 43, 301–304, http://dx.doi.org/10.1006/ stressors and stress responses: what commonalities appear between species
eesa.1999.1794. across biological organization levels? Environ. Pollut. 202, 66–77, http://dx.doi.
Sandrini-Neto, L., Camargo, M.G.m 2012. GAD: an R package for ANOVA designs org/10.1016/j.envpol.2015.03.013.
from general principles. R package version 1.1.1. Underwood, A.J., 1997. Experiments in Ecology: Their Logical Design Interpretation
http:/CRAN.R-project.org/package=GAD. Using Analysis of Variance. University Press, New York, Cambridge, pp. 504p.
Santos, C.S.G., Pleijel, F., Lana, P.C., Rouse, G.W., 2006. Phylogenetic relationships Ventura-Lima, J., Ramos, P.B., Fattorini, D., Regoli, F., Ferraz, L., Carvalho, L.M.,
within Nereididae (Annelida: Phyllodocida). Invertebr. Syst. 19, 557–576, Monserrat, J.M., 2011. Accumulation, biotransformation, and biochemical
http://dx.doi.org/10.1071/IS05001. responses after exposure to arsenate and arsenate in the estuarine polychaete
Schiedek, D., Broeg, K., Barsiene, J., Lehtonen, K.K., Gercken, J., Pfeifer, S., Laeonereis acuta (Nereididae). Environ. Sci. Pollut. Res. 18, 1270–1278, http://
Vuontisjarvi, H., Vuorinen, P.J., Dedonyte, V., Koehler, A., Balk, L., Schneider, R., dx.doi.org/10.1007/s11356-011-0478-4.
2006. Biomarker responses as indication of contaminant effects in blue mussel
(Mytilus edulis) and female eelpout (Zoarces viviparus) from the southwestern
Baltic Sea. Mar. Pollut. Bull. 53, 387–405, http://dx.doi.org/10.1016/j.
marpolbul.2005.11.013.
Smolders, R., Bervoets, L., De Coen, W., Blust, R., 2004. Cellular energy allocation in
zebra mussels exposed along a pollution gradient: linking cellular effects to
higher levels of biological organization. Environ. Pollut. 129, 99–112, http://dx.
doi.org/10.1016/j.envpol.2003.09.027.