Increased tolerance to oil exposure by the cosmopolitan marine copepod Acartia tonsa

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DOI: 10.1016/j.scitotenv.2017.06.139
Abstract
Oil contamination is an environmental hazard to marine ecosystems, but marine organism tolerance to oil after many generations of exposure remains poorly known. We studied the effects of transgenerational oil exposure on fitness-related traits in a cosmopolitan neritic copepod, Acartia tonsa. Copepods were exposed to an oil compound, the PAH pyrene at concentrations of 1, 10, 100 and 100 + (the saturated pyrene concentration in seawater) nM over two generations and measured survival, sex ratio, size at maturity, grazing rate and reproductive success. Exposure to the pyrene concentration of 100+ nM resulted in 100 % mortality before adulthood in the first generation. At the pyrene concentration of 100 nM, pyrene reduced the grazing rate, increased mortality, reduced the size of females and caused lower egg production and hatching success. Importantly, we found strong evidence for increased tolerance to pyrene exposure in the second generation: the reduction in size at maturity of females was less pronounced in the second generation and survival, egg production and hatching success were recovered to control levels in the second generation. The copepod increased tolerance to oil contamination may dampen the direct ecological consequences of a coastal oil spill, but it raises the concern whether a larger fraction of oil components accumulated in survived copepods may be transferred up the food web.
Increased tolerance to oil exposure by the cosmopolitan marine copepod
Acartia tonsa
Kamille Elvstrøm Krause
1
, Khuong V. Dinh
,1
, Torkel Gissel Nielsen
Section for Oceans and Arctic, National Institute of Aquatic Resources, Technical University of Denmark, Kemitorvet, bygning 201, Lyngby Campus, 2800 Kgs. Lyngby, Denmark
HIGHLIGHTS
New knowledge on the tolerance of ma-
rine organisms to oil exposure
The cosmopolitan copepod Acartia tonsa
was exposed to pyrene for two genera-
tions.
Pyrene (100nM) reduced survival,graz-
ing and egg production in the 1st gener-
ation.
Survival, egg production and hatching
success were recovered in the 2nd gen-
eration.
Second generation of Acartia tonsa
showed an increased tolerance to
pyrene exposure.
GRAPHICAL ABSTRACT
abstractarticle info
Article history:
Received 14 February 2017
Received in revised form 19 April 2017
Accepted 17 June 2017
Available online xxxx
Editor: Henner Hollert
Oil contamination is an environmental hazard to marine ecosystems, but marine organism tolerance to oil after
many generations of exposure remains poorly known. We studied the effects of transgenerational oil exposure
on tness-related traits in a cosmopolitan neritic copepod, Acartia tonsa. Copepods were exposed to an oil com-
pound, the PAH pyrene, at concentrations of 1, 10, 100 and 100+ (the saturated pyrene concentration in
seawater) nM over two generations and measuredsurvival, sex ratio, sizeat maturity, grazing rateand reproduc-
tive success. Exposure to the pyrene concentration of 100+ nM resulted in 100% mortality before adulthood in
the rst generation. At the pyrene concentration of 100 nM, pyrene reduced grazing rate, increased mortality, re-
duced the size of females and caused lower egg production and hatching success. Importantly, we found strong
evidencefor increased toleranceto pyrene exposure in the second generation: the reduction in size at maturityof
females was less pronounced in the second generation and survival, egg production and hatching success were
recovered to control levels in the second generation. The increased tolerance of copepods to oil contamination
may dampen the direct ecological consequences of a coastal oil spill, but it raises the concern whether a larger
fraction of oil components accumulated in survived copepods, may be transferred up the food web.
© 2016 Elsevier B.V. All rights reserved.
Keywords:
Coastal ecosystem
Egg production
Marine zooplankton
Oil spill
Polycyclic aromatic hydrocarbons
Transgenerational study
1. Introduction
Oil pollution from shipping and oil exploitation is a major potential
ecotoxicological hazard to marine ecosystems. It is well known that oil
spills such as Exxon Valdez or Deepwater Horizon causedimmediate ca-
tastrophes (Allan et al., 2012; Camilli et al., 2010; Joye, 2015; Peterson
et al., 2003). For example, the Exxon Valdez oil spill killed between
Science of the Total Environment 607608 (2017) 8794
Corresponding author.
E-mail address: kvdi@aqua.dtu.dk (K.V. Dinh).
1
Co-rst authors: K.E.K. and K.V.D.
http://dx.doi.org/10.1016/j.scitotenv.2017.06.139
0048-9697/© 2016 Elsevier B.V. All rights reserved.
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journal homepage: www.elsevier.com/locate/scitotenv
1000 and 2800 sea otters and approximately 250,000 seabirds
(Peterson et al., 2003). Both shipping and oil exploitation have been
predicted to increase in the near future to fulll the increasing global en-
ergy demand, which may in turn increase exposure of marine organ-
isms to crude oil worldwide (Barata et al., 2005; National Research
Council, 2003; Nørregaard et al., 2014). However, very little is known
about whether marine species, particularly those at the base of the
food web such as copepods, will develop an increased tolerance when
exposed to dissolved oil components e.g. polycyclic aromatic hydrocar-
bons PAHs (Reddy et al., 2012; Samanta et al., 2002).
Oil spills from shipping, oil seepage and offshore oil exploitations in-
troduce PAHs to the environment (Wolska et al., 2012) and PAHs are
toxic to marine animals (Almeda et al., 2013; Barata et al., 2005;
Incardona et al., 2012; Jensen et al., 2008). More than 20 different
PAHs have been found in coastal water affected by oil spills and pyrene
is one of the very common PAHs in crude oil (Reddy and Quinn, 2001;
Reddy et al., 2012). Pyrene has relatively low toxicity to marine organ-
isms compared to other PAHs (Barata et al., 2005). Furthermore, the
toxic effect of PAHs in mixture has been shown to be additive (Barata
et al., 2005), therefore the use of pyrene as a model PAH does not over-
estimate the toxicity of total PAHs (Jensen et al., 2008). The concentra-
tion of pyrene in coastal water varies from the trace level
(Annammala et al., 2013) to approximately 2.3 μgL
1
, equal to approx-
imately 12 nM (Reddy and Quinn, 2001), but the total PAH concentra-
tion can be up to 115 μgL
1
. The Deepwater Horizon oil spill released
approximately 387 tons of pyrene to the Gulf of Mexico (Reddy et al.,
2012), but there has not been given any clear information about the
concentration in the seawater.
There is evidence that aquatic animals may develop a tolerance to con-
taminants (e.g., Bach and Dahllof, 2012; Klerks et al., 2011; Morgan et al.,
2007; Ross et al., 2002) or toxins from phytoplankton (e.g., Colin and
Dam, 2002, 2005; Dam, 2013). The multigenerational experiment is a pow-
erful approach for detecting the development or expression of an increased
tolerance to contaminants or toxins across generations (e.g.
Carrera-Martinez et al., 2011; Colin and Dam, 2005; Romero-Lopez et al.,
2012). Recent studies have stressed the need for multigenerational ecotox-
icological studies to fully evaluate the effects of contaminants on the persis-
tence of natural populations (Kimberly and Salice, 2015; Perrichon et al.,
2015; Prud'homme et al., 2017). However, the potential for fast-growing
marine copepods with short life cycles to develop an increased tolerance
to PAHs across a transgenerational exposure has not been investigated.
In the coastal ecosystem, copepods play a key role as secondary pro-
ducers transferring energy up through the food web to the sh stock
(Kiørboe, 1998; Kwok et al., 2015). Any negative effects of oil exposure
on copepods may therefore cascade through the pelagic food web with
huge potential ecological and economic consequences. Copepods are
commonly used as model species in ecotoxicological studies of coastal
marine ecosystems (Kwok et al., 2015; Raisuddin et al., 2007). Exposure
to PAHs is known to reduce growth and fecundity and increase mortality
of marine copepods (Bellas and Thor, 2007; Hjorth and Nielsen, 2011;
Grenvald et al., 2013).
In this study, we tested the hypothesis that the short life cycled cope-
pod A. tonsa would develop an increased tolerance to pyrene exposure
after a continuous two-generation exposure. Fitness-related traits such
as mortality, size at maturity, sex ratio, grazing rate, egg production and
hatching success were quantied. We chose A. tonsa due to it being one
of the most abundant copepod species in nearshore marine environments
globally (Chen and Hare, 2008; Chen and Hares, 2011; Cervetto et al.,
1999; Paffenhöfer and Stearns, 1988; Pastorinho et al., 2003).
2. Materials and methods
2.1. Study species
Eggs of the copepod Acartia tonsa were obtained froma stock culture
at the National Institute of Aquatic Resources, Technical University of
Denmark. The culture of A. tonsa has been in the laboratory for
N30 years and is reared in 513 L black polyethylene tanks (h × d =
150 × 66 cm) lled with ca. 450 L ltered seawater. Temperature is sta-
ble at around 1618 °C (Støttrup et al., 1986) and salinity was kept sta-
ble at 32 ppt. The culture is provided with dim light which follows the
natural diel cycle (see more details in Drillet et al., 2008a). They are
fed ad libitum with the microalgae Rhodomonas salina (500 μgCL
1
)
(Berggreen et al., 1988). The density of the adult copepods in the stock
culture is maintained at approximately 100 individuals L
1
and the
eggs are harvested three times a week (Støttrup et al.,1986). Depending
on the temperature,it takes between 12 and 23 days for anegg to devel-
op to the adult stage (Finiguerra et al., 2013) and the lifespans of adult
males and females of A. tonsa vary from 10 to N40 days depending on
food availability (Finiguerra et al., 2013; Kiørboe et al., 2015). Each fe-
male can lay approximately 25 eggs per day on average (Støttrup
et al., 1986).
2.2. Filtered seawater and pyrene solutions
Seawater (salinity of 32 ppt) was ltered through a two-time 0.2 μm
ltering system. Water was kept in the laboratory for 24 h prior to ex-
perimental use to stabilize the temperature. pH was 88.2 and the dis-
solved oxygen was approximately 7 mg L
1
.
Two stock solutions of 0.2 (stock 1) and 1 mM (stock 2) were made
by dissolving pyrene powder (Sigma-Aldrich, purity N99%) in absolute
acetone. Both stock solutions were kept in amber glass bottles wrapped
in aluminum foil to avoid photodegradation of pyrene. Stock solutions
were kept at a room temperature of approximately 20 °C. The exposure
solutions were daily prepared by diluting the stock solutions. Stock 1
was diluted 5000 and 500 times in ltered seawater to prepare the ex-
posure solutions of 1 and 10 nM, respectively. Similarly, stock 2 was di-
luted 10,000 and 3333 times in ltered water to obtain the exposure
solutions of 100 and 100+ nM, respectively. The concentration of ace-
tone in the acetone control was 300 μlL
1
, which was equal to the ace-
tone concentration in the highest pyrene exposure solution (100+ nM).
The laboratory was kept dark during the experiment as the toxicity of
pyrene substantially increases when exposed to sunlight (Pelletier
et al., 1997).
2.3. Pyrene exposure experiment
To test whether copepods develop an increased tolerance to oil ex-
posure a transgenerational experiment was conducted in which
A. tonsa was exposed to six different exposure solutions: seawater con-
trol, acetone solvent control, 1, 10, 100 and 100+ (saturated pyrene
concentration in seawater, Nørregaard et al., 2014) nM pyrene for two
generations. The pyrene concentrations were chosen based on previous
studies showing negative effects on the performance of marine cope-
pods (Jensen et al., 2008; Grenvald et al., 2013; Nørregaard et al.,
2014). The highest pyrene concentration in this study (100+ nM,
equal to approximately 90 μgL
1
) was lower than the concentration
of total PAHs, of 115 μgL
1
measured in seawater affected by oil spills
(Reddy and Quinn, 2001). The measured pyrene concentrations in ex-
posure solutions (based on pooled samples collected from 5 different
bottles per concentration) were 0.7, 8, 164 and 457 nM when the medi-
um was freshly renewed. After 24 h, the real exposure concentrations
were 3, 7, 152 and 415 nM, respectively. An independent research
group (Lovap, Belgium) performed all measurements of pyreneconcen-
trations. The acetone control was included in the experiment as acetone
was used as a solvent for pyrene. An acetone concentration of up to 900
μlL
1
(three times higher than the highest acetone concentration used
in this experiment) has no effecton survivaland fecal pellet production
in another calanoid copepod, Calanus nmarchicus (see Appendix A).
Each treatment had ve replicates (a total of 30 experimental units for
the rst generation and 25 experimental units for the second
generation).
88 K.E. Krause et al. / Science of the Total Environment 607608 (2017) 8794
To start thepyrene exposure experiment eggs fromthe stockculture
were collected, and mixed with 1 L of sea water (32 ppt) to create the
stock solution of eggs. The density of eggs in this stock solution was de-
termined by subsampling 10 mL of solution by a Kip dispenser head
(10 mL, Duran, Germany), pouring the content into a Petri dish,
counting the number of eggs under a microscope (SZ40, Olympus,
Tokyo, Japan), and then calculating the density. Based on the density
of eggs in the stock solution, approximately 301 ± 15 eggs (mean ±
SE, n = 30 counts from 30 experimental bottles) were added to each ex-
perimental bottle by the Kip dispenser head. Experimental units were
acid cleaned glass bottles (volume of 1.12 L, with thermoplastic polyes-
ter cap and PTFE sealing disc) lled with the exposure solutions and an
algal concentration of approximately 20,000 cells mL
1
(equivalent to
948 μgCL
1
)(Berggreen et al., 1988). All bottles were lled up to the
brim and closed with the described cap to avoid air bubbles. They
were then mounted on a plankton wheel (0.5 r min
1
). Temperature
was monitored in two water lled bottles using thermo loggers. The ex-
perimental media with exposure solutions and the algal concentration
were renewed daily. In the rst generation the adult stage was observed
after 1112 days by daily observing copepods under microscope during
the medium refreshment. Copepods used for checking the developmen-
tal stages were collected by ltration from two extra bottles, that were
set up in parallel withthe experimentalbottles, one with clean seawater
and another with a 100 nM pyrene exposure solution. On day 14, two to
ve females from each bottle were collected for testing fecal pellet and
egg production (see more in Section 2.4). The rest of the copepods
remained in the bottles for 24 h to produce eggs for the testing of hatch-
ing success and to start the second generation. Eggs were isolated by l-
tration from each respective experimental bottle and divided into two
subsamples, one for hatching success and a similar amount of eggs
was used to initiate the second generation. Approximately 526 ± 41
eggs (mean ± SE, n = 25 counts for 25 experimental bottles) were
added to each of the respective bottles to start the second generation.
Note that the stocking densities used in this study were lower than
the 1000 eggs L
1
used by Franco et al. (2017), which showed no effect
on mortality, growth and development of A. tonsa. The exposure exper-
iment for the second generation and the observation of the develop-
ment of copepods were repeated as in the rst generation. The
duration of the whole exposure experiment lasted 29 days covering
two generations.
We also tested if exposure to pyrene affected algal density, thereby
altering the density of algae available as food for copepods. Similarly,
we also tested whether exposure to pyrene resulted in changed hatch-
ing success of eggs from the same stock culture batch as the eggs used
for the main exposure experiment, to account for varying initial density
of copepods. There were no differences in algal densities (Fig. S2, Ap-
pendix A) or hatching success (Fig. S3, Appendix A) between the two
controlsand the pyrene concentrations(see more detail in Appendix A).
2.4. Response variables
The number of live adult copepods was counted on day 15 and30 for
the rst and second generations, respectively. Survival was calculated as
the percentage of initial eggs of each generation surviving to maturity.
The sex ratio was dened asthe number of males over the number offe-
males in each experimental unit. The prosome lengths (μm) of adult
males and females were measured and were converted to biomass
using the regression (Berggreen et al., 1988):
W¼1:11 105L2:92
;
where W is body weight in ng C (converted to μgCinthenal analyses)
and L is prosome length in μm.
Fecal pellet (proxy for grazing activity in copepods, Besiktepe and
Dam, 2002; Isla et al., 2008) and egg production were assessed for
each of the two generations at all exposure solutions in ve replicates
by randomly collecting two to ve females from each experimental bot-
tle on day 14 and 29 for th e rst and secondgeneration, respectively. Fe-
males were transferred with a pipette to acid cleaned glass bottles
(volume of 0.25 L, with thermoplastic polyester cap and PTFE sealing
disc) containing the same medium and water treatment as the bottle
they originated from. The bottles were incubated for 24 h on the plank-
ton wheel. Fecal pellets and eggs were poured into a lter (mesh size =
30 μm) and the content was carefully rinsed into a Petri dish. The num-
ber of fecal pellets and eggs in each petri dish was counted under a mi-
croscope (SZ40, Olympus, Tokyo, Japan). The egg production was
estimated as eggs female
1
d
1
and the fecal pellet production was es-
timated as fecal pellets female
1
d
1
.
2.5. Hatching success
Two hatching tests were conducted over 48 h in the same exposure
media as the exposure experiment, one for each generation. Eggs used
for the hatching test for the rst generation was the same eggs used to
initiate thesecond generation (see Section 2.3). Eggs used for the hatch-
ing test of the second generation were concentrated from each respec-
tive experimental bottle by ltration. The media were refreshed after
24 h. The hatching success wasestimated as the percentage of the initial
number of eggs hatched to nauplii after 48 h.
2.6. Statistical analyses
To test for the effectsof pyrene exposureon survival of different gen-
erations, we ran a general linear model (GLM) (see Warton and Hui,
2011) with pyrene and generation as xed factors. We also ran similar
GLMs for other response variables including sex ratio, size at maturity,
fecal pellet production, egg production, and hatching success. For size
at maturity we included sex as a xed factor in the GLMs, as the sensi-
tivity of copepods to pyrene may besex-specic, as shown in a previous
study with pyrethroid cypermethrin (Medina et al.,2002). Note that it is
not possible to identify the sex ofcopepods prior to adulthood, whereas
the assessment of survival rate for separated sexes was not possible. For
all GLMs, we tested the assumption of normality of the error distribu-
tions with Shapiro-Wilk tests and the homogeneity of variances with
Levene's tests. When the homogeneity of variances (survival, egg pro-
duction) was not met, data were log(x + 1)-transformed to meet
model assumptions. Statistical differences were considered signicant
if Pb0.05. All statistical analyses were done in Statistica 12 (StatSoft
Inc., Tulsa, OK, United States). Data are presented in the gures as
Least-square means + SEs.
3. Results
3.1. Survival
At low pyrene concentrations of 110 nM mean survival varied from
56 to 76% and did not differ from controls or between the rst and the
second generation (contrast analyses of the rst vs. second generation
at: 1 nM, F
1, 40
=2.00,P=0.17and10nMF
1, 40
=2.68,P=0.11,
Fig. 1). At the pyrene concentration of 100 nM survival was strongly re-
duced to approximately 28% in the rst generation (contrast analysis
between seawater control vs. 100 nM of the rst generation, F
1, 40
=
50.08, Pb0.001), but increased to approximately 57% in the second gen-
eration, which did not differ from the survival of the control (71% in the
second generation) (contrast analysis between seawater control vs.
100 nM of the second generation F
1, 40
=2.72,P= 0.11), generating a
pyrene × generation interaction (Table 1,Fig. 1). Exposure to the con-
centration of saturated pyrene in seawater (100+ nM) resulted in
100% mortality before adulthood in the rst generation (Fig. 1).
89K.E. Krause et al. / Science of the Total Environment 607608 (2017) 8794
3.2. Sex ratio
Exposure to pyrene had no effect on the sex ratio of A. tonsa
(Table 1). The ratio was however higher in the rst generation com-
pared to the second generation (Table 1,Fig. 2). This pattern did not de-
pend on pyrene exposure.
3.3. Size and biomass at maturity
Exposure to pyrene concentrations of 1 and 10 nM had no effect on
the size and biomass of males or females at maturity (Fig. 3ad). Expo-
sure to 100 nM pyrene resulted in reduced size and biomass of females,
but not males (Pyrene × sex, Table 2,Fig. 3ad). The pyrene-induced re-
duction in size of females in the second generation (female size reduc-
tion of 4%; female biomass reduction of 11%) was less than half of that
of the rst generation (female size reduction of 11% and female biomass
reduction of 28%) (Pyrene × generation × sex, Table 2,Fig. 3b, d).
3.4. Fecal pellet production
Fecal pellet production (proxy of grazing rate) was higher in the ac-
etone control compared to the seawater control and the 10 and 100 nM
treatments (Duncan Post-hoc test, Pb0.04), but no difference was de-
tected between pyrene concentrations and seawater control (Duncan
Post-hoc tests, all Pvalues N0.17, Fig. 4a). Fecal pellet production was
lower in the second generation compared to the rst generation
(main effect Generation, Table 3,Fig. 4a); and this pattern was consis-
tent in all pyrene concentrations.
3.5. Egg production
Overall, exposure to pyrene tended to reduce egg production
(Table 3,Fig. 4b). There was no difference in egg production between
the two generations and there was no interaction between pyrene expo-
sure and generation (P= 0.55). Egg production covaried positively with
the fecal pellet production (F
1, 39
= 2.04, P= 0.048, slope ± 1 SE =
0.0044 ± 0.0022).
3.6. Hatching success
At the pyrene concentrations of 1 and 10 nM, the hatching success of
eggs from the rst and second generation was generally N90% and did
not differ signicantly from the 8090% hatching success in the controls
(all Pvalues N0.059). Exposure to the pyrene concentration of 100 nM
strongly reduced hatching success of eggs from the rst generation to
approximately 50%, but the hatching success of eggs from the second
generation was equal to the control (seawater control = 99 ± 0.19%
and 100 nM = 94 ± 3.42%, contrast analysis F
1, 40
= 0.86, P= 0.36)
(Table 3,Fig. 4c). The hatching success of the seawater control was
higher in the rst generation (80.52 ± 7.02%) compared to the second
generation (99.37 ± 0.19%) (contrast analysis F
1, 40
=11.84,P=
0.0013).
4. Discussion
Copepods account for N80% of the mesozooplankton biomass in the
marine food web (Kiørboe, 1998), any effects of oil exposure on cope-
pods may cascade through the food chains causing great ecological
and economic effects. Here, we document strong evidence for an in-
creased tolerance of the copepod Acartia tonsa in the second generation
after continuous exposure to pyrene, a component of crude oil toxic to
marine animals (see e.g. Nørregaard et al., 2014). In the following sec-
tion, we will discuss the effects of pyrene exposure on tness-related
traits of copepods, the sex-specic response and then the difference in
sensitivity of A. tonsa between generations.
4.1. Pyrene effects
There is extensive evidence that exposure to crude oil substances re-
duces the survival in marine organisms, e.g. scyphozoans and cteno-
phores (Almeda et al., 2013), shes (Heintz et al., 1999; Hicken et al.,
2011; Incardona et al., 2012) and also copepods (Bellas and Thor,
2007; Grenvald et al., 2013). Accordingly, the survival of A. tonsa was
strongly reduced at the pyrene concentrations of 100 and 100+ nM in
the rst generation. The pyrene-induced mortality has been suggested
as a result of a narcotization resulting in reduced feeding, which can ul-
timately lead to death (Jensen et al., 2008). Our results supported this
suggestion, as the fecal pellet production was lower in copepods ex-
posed to 100 nM than in the acetone control. Pyrene also caused a re-
duction in size and biomass of females, which may also be the result
of reduced feeding together with the higher energy investment in de-
toxication and defense systems against pyrene. Previous studies have
Fig. 1. Survival of the copepodAcartia tonsa as a function of pyrene e xposure conce ntration
and generation. Data are least-square means + 1 SE.
Table 1
The results of general linear models testing for the effects of pyrene exposure on survival
and sex ratio ofthe copepod Acartia tonsaacross two generations. Signicant Pvalues(Pb
0.05) are indicated in bold.
Effects Survival Sex ratio
df1, df2 F Pdf1,df2 F P
Pyrene 4, 40 13.93 b0.001 4, 40 0.26 0.90
Generation 1, 40 0.038 0.85 1, 40 8.65 0.0054
Pyrene × generation 4, 40 7.45 b0.001 4, 40 0.26 0.90
Fig. 2. Ratio of males to fem ales of the copepod Acartia tonsa as a function of pyrene
exposure concentration and generation. Data are least-square means + 1 SE.
90 K.E. Krause et al. / Science of the Total Environment 607608 (2017) 8794
shown that exposure to pyrene can result in the upregulation of detox-
ication enzymes e.g., CYP1A and AhR2 in sh species (Hicken et al.,
2011; Incardona et al., 2005) and increased activity of antioxidant en-
zymes such as glutathione S-transferase, glutathione reductase, and cat-
alase in the marine copepod Tigriopus japonicas (Han et al., 2014).
Whatever mechanisms, the size of females is a major determinant of re-
productive success in copepods (Kiørboe and Sabatini, 1995; Sichlau
and Kiørboe, 2011), and a reduced size in pyrene-exposed females
may therefore have ecological consequences (Sichlau and Kiørboe,
2011).
Reproductive success is one of the key tness traits in organisms and
a reduction in reproductive success may translate into a population de-
cline. In our study, the overall egg production and hatching success of
A. tonsa eggs from controls to 10 nM of pyrene treatments were high
(8090%). This pattern was in agreement with a previous study show-
ing that when the egg production is high (20 eggs female
1
day
1
),
then the hatching success is also high (80100%) (Tang and Dam,
2001). This was also true for the hatching success of eggs collected
from 100 nM in the second generation (Fig. 4b, the last bar).
The reduced egg production in the 100 nM treatment in the rst
generation can be explained by the reduced feeding. It is well known
that A. tonsa egg production depends on grazing rate (Kiørboe et al.,
1985), we also found a positive correlation between egg and fecal pellet
production. Yet, this correlation cannot alone explain the recovery of
egg production in the second generation since the fecal pellet produc-
tion was relatively lower in the rst generation. Possibly, the copepods
may have prioritized the energy intake to increase thereproductive out-
put as a compensatory mechanism in response to increased mortality in
the rst generation.
The reduced hatchingsuccess of eggs collected from 100 nM pyrene
in the rst generation was in agreement with the pattern observed in
previous studies exposing females to pyrene and other PAHs (Bellas
and Thor, 2007; Hjorth and Nielsen, 2011; Jensen et al., 2008;
Nørregaard et al., 2014). The eggshell of copepod eggs has been
shown to act as an efcient protection against external contaminants
(Charmantier and Charmantier-Daures, 2001; Grenvald et al., 2013;
Sichlau et al., 2011) or toxins from toxic algae (Tang and Dam, 2001)
and is impermeable to PAHs (Jensen and Carroll, 2010). Therefore, the
observed low hatching success was unlikely a result of direct exposure
to pyrene, but more likely a result of thecarryover effect from intoxicat-
ed females to their eggs. This was supported by the fact that hatching
Fig. 3. Sizeand biomass of males(a, c) and females (b, d) ofthe copepod Acartia tonsa as a function of pyreneexposure concentration and generation. Dataare least-square means+ 1 SE.
Table 2
The results of general linear models testing for the effects of pyrene exposure on size and
biomass at maturity of males and females of the copepod Acartia tonsa across two gener-
ations. Signicant Pvalues (Pb0.05) are indicated in bold.
Effects Size at maturity Biomass at maturity
df1,
df2
FPdf1,df2 F P
Pyrene 4, 80 38.63 b0.001 4, 80 47.29 b0.001
Generation 1, 80 2.51 0.12 1, 80 2.01 0.16
Sex 1, 80 3566.60 b0.001 1, 80 3897.10 b0.001
Pyrene × generation 4, 80 5.83 b0.001 4, 80 5.95 b0.001
Pyrene × sex 4, 80 34.73 b0.001 4, 80 45.70 b0.001
Generation × sex 1, 80 6.69 0.012 1, 80 6.63 0.012
Pyrene × generation × sex 4, 80 7.68 b0.001 4, 80 8.69 b0.001
91K.E. Krause et al. / Science of the Total Environment 607608 (2017) 8794
success of the eggs from the stock culture was not lower in the pyrene
treatments compared to the control (see Appendix A). Exposed females
may accumulate pyrene in their lipid stores as has been shown previ-
ously in many marine organisms exposed to PAHs (Berrojalbiz et al.,
2009; Jensen et al., 2012; Nørregaard et al., 2015). The accumulated
toxins and their metabolites may have a negative effect on the matura-
tion of the egg and embryonic development as it has been shown on the
vitellogenesis of marine sh (Nicolas, 1999). The transmissions of PAHs
such as benzo(α)pyrene from mothers to offspring has been observed
in athead soles. In the case of the athead soles, exposed sh produced
oocytes and semen with high level of benzo(α)pyrene and its metabo-
lites and the hatching success of intoxicated eggs was ve times lower
than the control (Hose et al., 1981). We also found a statistically signif-
icant lower hatching success in the seawater control of the rst genera-
tion compared to the second generation, but the hatching success of
both generations were within the normal range of A. tonsa originating
from the same culture (7599%, strain DIFRES, Fig. 4)(Drillet et al.,
2008b).
4.2. Sex-speciceffects
Sex-specic vulnerability to contaminants and toxins is well-known
in aquatic species (Campero et al., 2008; Dinhet al., 2016), including co-
pepods (e.g., Avery et al., 2008; Medina et al., 2002). We found a differ-
ence in the response of males and females to pyrene exposure, but the
sex-specic vulnerable pattern was not in the same direction as what
have been observed in a related species A. fundyense exposed to toxic
algae (Avery et al., 2008). In the study of Avery et al. (2008), naïve cope-
pod A. fundyense males were more sensitive, succumbing at higher rates
to the presence of algal toxins than females while in our study male co-
pepods showed no size difference between pyrene treatments and the
controls, but female length was reduced by 411% at the pyreneconcen-
tration of 100 nM compared to the controls. It has been known that
males of A. tonsa spend less time feeding compared to females
(Kiørboe, 2007) and therefore might ingest less pyrene, as feeding is
suggested to be a major route of pyrene absorption in copepods
(Jensen et al., 2008).
4.3. Generational effect and the increased tolerance to oil exposure
The male to female ratio was also higher in the second generation
compared to the rst generation. A previous study has suggested that
the differential mortality caused by toxic algae resulted in the skewed
sex ratios towards more females (Avery et al., 2008). However, our re-
sult showing the higher ratio of males to females in the rst generation
compared to the second generation could just be due to chance as this
pattern did not depend on pyrene exposure, as it was consistent from
controls to the 100 nM treatment. In copepods, the sex ratio can vary
greatly and cannot fully be explained by the different longevity or mor-
tality during the development (Gusmao and McKinnon, 2009).
The most important ndings of the current study were three lines of
evidence supporting an increased tolerance to pyrene exposure in the
second generation: survival and hatching success were recovered to
control levels and the reduction in female size was less pronounced
compared to the rst generation. An increase in tolerance of aquatic an-
imals after long-term exposure to contaminants, toxins or climatic
stressors has previously been observed in marine zooplankton species
exposed to toxic algae (Colin and Dam, 2002, 2005) and to ocean acidi-
cation (Thor and Dupont, 2015), but this is, surprisingly, the rst time
it has been documented for copepods exposed to a PAH. The develop-
ment of an increased tolerance to stressors across generations may be
a result of developmentalplasticity, maternal effects orgenetic selection
Fig. 4. Fecal pellet (a) and egg production (b) and the hatching success (c) of the copepod
Acartia tonsa as a function of pyrene exposure concentration and generation. Data are
least-square means + 1 SE.
Table 3
The results ofgeneral linear models testing for the effects of pyrene exposure on the fecal
pellet and egg production of females and the hatching success of eggs of the copepod
Acartia tonsa across two generations. Signicant Pvalues (Pb0.05) are indicated in bold.
Effects Fecal pellets Egg production Hatching success
df1,
df2
FPdf1,
df2
FPdf1,
df2
FP
Pyrene 4, 40 3.19 0.023 4, 40 2.46 0.061 4, 40 10.99 b0.001
Generation 1, 40 6.79 0.013 1, 40 0.005 0.94 1, 40 39.99 b0.001
Pyrene ×
generation
4, 40 1.45 0.24 4, 40 0.78 0.55 4, 40 9.49 b0.001
92 K.E. Krause et al. / Science of the Total Environment 607608 (2017) 8794
(Robertson et al., 2017). The high mortality (72% mortality from eggs to
adults) found when copepods were exposed to 100 nM in the rst gen-
eration may suggest some kind of genetic selection in the A.tonsa pop-
ulations, but we could not rule out the role of developmental plasticity
or maternal effects as the current experimental design did not allow
partitioning of the plasticity and the changes in genetic viability of
A. tonsa in contributing to the increased tolerance to pyrene. If the in-
creased tolerance to pyrene here had a genetic basis, then this evolution
of adaptive response may have come with a cost (reviewed in Dam,
2013) such as reducing the genetic diversity (Gardeström et al., 2008;
Ross et al., 2002) or reducing their ability to deal with other important
stressors such as climate change (Moe et al., 2013).
Oil contaminantion is a major concern worldwide (Hicken et al.,
2011) and serious impacts of oil spills on marine ecosystems have
been documented (Incardona et al., 2012, 2014; Peterson et al., 2003;
Paine et al., 1996). Pyrene is a very common PAH and always present
in water contaminated with crude oil. The concentration of PAHs in
coastal water affected by shipping is often at lower levels
(Annammala et al., 2013). However, in coastal water affected by oil
spills the concentration of total PAHs, including pyrene can be at ex-
tremely high levels. For example, a concentration of 115 μgL
1
for
total PAHs has been measured in coastal water off Rhode Island affected
by the North Cape oil spill (Reddy and Quinn, 2001).
There is a concern that the use of laboratory-reared copepod A. tonsa,
which has been cultured for N30 years in a stable and clean environ-
ment, may have lower standing genetic diversity than that of natural
populations, thereby making the extrapolation of the observed effects
non-compatible with natural ecosystems. While we partly agree that a
decrease in genetic diversity during such a long isolation period is likely
inevitable, this process should be very slow given the high number of
copepods in the stock culture, which are approximately 45,000 individ-
uals per tank. This is far more than the number of 5001000 individuals
for A. tonsa suggested to avoid inbreeding (Colin and Dam, 2005). Fur-
thermore, the laboratory culture and wild-collected A. tonsa have
shown both similar mortality patterns and swimming activity in re-
sponse to the presence of food (Tiselius et al., 1995).
The current results provide evidence for the potential of an increased
toleranceto oil exposure by fast growingmarine animals with short life-
cycles like thecopepod A. tonsa. The high resilience of coastal organisms
in dealing with oil spills has been shown in other species such as Spar-
tina alterniora (Robertson et al., 2017). As copepods are key species
in coastal and marine ecosystems (Chen and Hare, 2008; Chen and
Hares, 2011; Kiørboe, 1998; Kwok et al., 2015; Paffenhöfer and
Stearns, 1988), the increased tolerance to oil exposure may reduce the
direct ecological consequences of the pressence of dissolved oil compo-
nents in the environment. Future studies should partition the contribu-
tion of plasticity and genetic basis to the increased tolerance and the
related costs. Irrespective of the mechanisms for the recovery of
A. tonsa tness-related traits despite pyrene exposure in the second
generation, it may dampen the direct ecological consequences of a
coastal oil spill. Although, it also raises the concern whether ot not the
survivingcopepods may accumulate greater quantities of toxic oil com-
ponents, which could then be transferred up through the food web
(Dam, 2013).
4.4. Conclusions
The occurrence of oil contamination at low concentrations is expect-
ed to be widespread in the future as a result of greater shipping activity
and expanding oil exploitations (National Research Council, 2003). It is
therefore especially relevant to test the ability of marine organisms to
deal with the changing conditions. Our results highlight the importance
of multigenerational studies inecotoxicology (e.g. Colin and Dam, 2005;
Kimberlyand Salice, 2015; Klerks et al., 2011) to fully capture the effects
of oil exposure on marine copepods in biologically diverse and produc-
tive coastal ecosystems.
Acknowledgements
We thank two anonymous reviewers for their constructive com-
ments that improved the manuscript. This work was nancially sup-
ported by Ørsted Fellowship to Khuong Van Dinh and by the
Norwegian Research Council under project no. 243923/E40 to Torkel
Gissel Nielsen. We thank Dr. Mary S. Wisz and Dr. Lis Bach for construc-
tive comments and proofreading, and Vu Thi Thuy Minh and Jack
Melbye for technical assistance during the experiment. The photo of
an Acartia tonsa female for the graphical abstract is credited to Dr. Vu
Thi Thuy Minh.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2017.06.139.
References
Allan, S.E.,Smith, B.W., Anderson, K.A., 2012. Impact of the Deepwater Horizon Oil Spill on
bioavailable polycyclic aromatic hydrocarbons in Gulf of Mexico coastal waters. Envi-
ron. Sci. Technol. 46, 20332039.
Almeda, R., Wambaugh, Z., Chai, C., Wang, Z., Liu, Z., Buskey, E.J.,2013. Effects of crude oil
exposure on bioaccumulation of polycyclic aromatic hydrocarbons and survival of
adult and larval stages of gelatinous zooplankton. PLoS One 8.
Annammala, K.V., Abdullah, M.H., Bin Mokhtar, M., Joseph, C.G., Sakari, M., 2013. Charac-
terization of aromatic hydrocarbons in tropical coastal water of Sabah, Borneo. Asian
J. Chem. 25, 37733780.
Avery, D.E., Altland, K.K., Dam, H.G., 2008. Sex-related differential mortality of a marine
copepod exposed to a toxic dinoagellate. Limnol. Oceanogr. 53, 26272635.
Bach, L., Dahllof, I., 2012. Local contamination in relation to population genetic diversity
and resilience of an arctic marine amphipod. Aquat. Toxicol. 114, 5866.
Barata, C., Calbet, A., Saiz, E., Ortiz, L., Bayona, J.M., 2005. Predicting single and mixture
toxicity of petrogenic polycyclic aromatic hydroc arbons to the copepod Oithona
davisae. Environ. Toxicol. Chem. 24, 29922999.
Bellas, J., Thor, P., 2007. Effects of selected PAHs on reproduction and su rvival of the
calanoid copepod Acartia tonsa. Ecotoxicology 16, 465474.
Berggreen,U., Hansen, B., Kiørboe, T., 1988. Food size spectra, ingestion and growth of the
copepod Acartia tonsa during development: implications for the determination of co-
pepod production. Mar. Biol. 99, 341352.
Berrojalbiz, N., Lacorte, S., Calbet, A., Saiz, E., Barata, C., Dachs, J., 2009. Accumulation and
cycling of polycyclic aromatic hydrocarbons in zooplankton. Environ. Sci. Technol. 43,
22952301.
Besiktepe,S., Dam, H.G., 2002. Coupling of ingestion and defecation as afunction of diet in
the calanoid copepod Acartia tonsa. Mar. Ecol. Prog. Ser. 229, 151164.
Camilli, R., Reddy, C.M., Yoerger, D.R., Van Mooy, B.A.S., Jakuba, M.V., Kinsey, J.C., et al.,
2010. Tracking hydrocarbon plume transport and biodegradationat Deepwater Hori-
zon. Science 330, 201204.
Campero, M., De Block, M., Ollevier, F., Stoks, R., 2008. Correcting the short-term effect of
food deprivation in a damsely: mechanisms and costs. J. Anim. Ecol. 77, 6673.
Carrera-Martinez, D., Mateos-Sanz, A., Lopez-Rodas, V., Costas, E., 2011. Adaptation of
microalgae to a gradient of continuous petroleum contamination. Aquat. Toxicol.
101, 342350.
Cervetto,G., Gaudy, R., Pagano, M., 1999.Inuence of salinityon the distribution of Acartia
tonsa (Copepoda, Calanoida). J. Exp. Mar. Biol. Ecol. 239, 3345.
Charmantier, G., Charmantier-Daures, M., 2001. Ontogeny of osmoregulation in crusta-
ceans: the embryonic phase. Am. Zool. 41, 10781089.
Chen, G., Hare, M.P., 2008. Cryptic ecological diversication of a planktonic estuarine co-
pepod, Acartia tonsa.Mol. Ecol. 17, 14511468.
Chen,G.,Hares,M.P.,2011.Cryptic diversity and comparative phylogeography
of the estuarine copepod Acartia tonsa ontheUSAtlanticcoast.Mol.Ecol.
20, 24252441.
Colin, S.P.,Dam, H.G., 2002. Latitudinal differentiation inthe effects of the toxic dinoagel-
late Alexandrium spp. on the feeding and reproduction of populations ofthe copepod
Acartia hudsonica. Harmful Algae 1, 113125.
Colin, S.P., Dam, H.G., 2005. Testing for resistance of pelagic marine copepods to a toxic
dinoagellate. Evol. Ecol. 18, 355377.
Dam, H.G., 2013. Evolutionary adaptation of marine zooplankton to global change. Annu.
Rev. Mar. Sci. 5, 349370.
Dinh, K.V., Janssens, L., Therry, L., Bervoets, L., Bonte, D., Stoks, R., 2016. Delayed effects of
chlorpyrifos across metamorphosis on dispersal-related traits in a poleward moving
damsely. Environ. Pollut. 218, 634643.
Drillet, G., Goetze, E., Jepsen, M., Hojgaard, J.K., Hansen, B.W., 2008a. Strain-specicvital
rates in fourAcartia tonsa cultures, I: strainorigin, genetic differentiation and egg sur-
vivorship. Aquaculture 280, 109116.
Drillet, G., Jepsen, P.M., Hojgaard, J.K., Jorgensen, N.O.G., Hansen, B.W., 2008b. Strain-
specic vital rates in four Acartia tonsa cultures II: life history traits and biochemical
contents of eggs and adults. Aquaculture 279, 4754.
Finiguerra, M.B., Dam, H.G., Avery, D.E., Burris, Z., 2013. Sex-specic tolerance to starva-
tion in the copepod Acartia tonsa.J.Exp.Mar.Biol.Ecol.446,1721.
93K.E. Krause et al. / Science of the Total Environment 607608 (2017) 8794
Franco, S.C., Augustin, C.B., Geffen, A.J., Dinis, M.T., 2017. Growth, egg production and
hatching success of Acartia tonsa cultured at high densities. Aquaculture 468 (Part
1), 569578.
Gardeström, J., Dahl, U., Kotsalainen, O., Maxson, A., Elfwing,T., Grahn, M., et al., 2008. Ev-
idence of population genetic effects of long-term exposure to contaminated sedi-
ments - a multi-endpoint study with copepods. Aquat. Toxicol. 86, 426436.
Grenvald, J.C., Nielsen, T.G., Hjorth, M., 2013. Effects of pyrene exposure and temperature
on early development of twoco-existing arcticcopepods. Ecotoxicology 22, 184198.
Gusmao, L.F.M., McKinnon, A.D., 2009. Sex ratios, intersexuality and sex change in cope-
pods. J. Plankton Res. 31, 11011117.
Han, J., Won, E.-J., Hwang, D.-S., Shin, K.-H., Lee, Y.S., Leung, K.M.-Y., et al., 2014. Crude oil
exposure results in oxidative stress-mediated dysfunctional development and repro-
duction in the copepod Tigriopus japonicus and modulates expression of cytochrome
P450 (CYP ) genes. A quat. Tox icol. 152, 308317.
Heintz, R.A., Short, J.W., Rice, S.D., 1999. Sensitivity of sh embryos to weathered crude
oil: part II. Increased mortality of pink salmon (Oncorhynchus gorbuscha) embryosin-
cubating downstream from weathered Exxon Valdez crude oil . Environ. Toxicol.
Chem. 18, 494503.
Hicken, C.E., Linbo, T.L., Baldwin, D.H., Willis, M.L., Myers, M.S., Holland, L., et al., 2011.
Sublethal exposure to crude oil during embryonic development alters cardiac mor-
phology and reduces aerobic capacity in adult sh. Proc. Natl. Acad. Sci. U. S. A. 108,
70867090.
Hjorth, M., Nielsen, T.G., 2011. Oil exposure in a warmer Arctic: potential impacts on key
zooplank ton species. Mar. Biol. 158, 13391347.
Hose, J.E.,Hannah, J.B., Landolt, M.L., Miller, B.S., Felton, S.P., Iwaoka, W.T., 1981. Uptake of
benzo(α)pyrene by gonadal ti ssue of atsh (family Pleuronectidate) and its effects
on subsequent egg development. J. Toxicol. Environ. Health 7, 9911000.
Incardona, J.P., Carls, M.G., Teraoka, H., Sloan, C.A., Collier, T.K., Scholz, N.L., 2005. Aryl hy-
drocarbon receptor-independenttoxicity of weathered crude oil during sh develop-
ment. Environ. Health Perspect. 113, 17551762.
Incardona, J.P., Gardner, L.D., Linbo, T.L., Brown, T.L., Esbaugh, A.J., Mager, E.M.,et al., 2014.
Deepwaterhorizon crude oil impacts the developing hearts of large predatorypelagic
sh. Proc. Natl. Acad. Sci. U. S. A. 111, E1510E1518.
Incardona, J.P., Vines, C.A., Anulacion, B.F.,Baldwin, D.H., Day,H.L., French, B.L., et al., 2012.
Unexpectedly high mortality in Pacic herring embryos exposed to the 2007 Cosco
Busan oil spill in San Francisco Bay. Proc. Natl. Acad. Sci. U. S. A. 109, E51E58.
Isla, J.A., Lengfellner, K., Sommer, U., 2008. Physiological response of the copepod
Pseudocalanus sp. in the Baltic Sea at different thermal scenarios. Glob. Chang. Biol.
14, 895906.
Jensen, L.K., Carroll, J., 2010. Experimental studies of reproduction and feeding for two
Arctic-dwelling Calanus species exposed to crude oil. Aquat. Biol. 10, 261271.
Jensen, L.K., Honkanen, J.O., Jaeger, I., Carroll, J., 2012. Bioaccumulation of phenanthrene
and benzo a pyrene in Calanus nmarchicus. Ecotoxicol. Environ. Saf. 78, 225231.
Jensen, M.H., Nielsen, T.G., Dahlloef, I., 2008. Effects of pyrene on grazing and reproduc-
tion of Calanus nmarchicus and Calanus glacialis from Disko Bay, West Greenland.
Aquat. Toxicol. 87, 99107.
Joye, S.B., 2015. Deepwater horizon, 5 years on. Science 349, 592593.
Kimberly, D.A., Salice, C.J., 2015. Multigenerational contaminant exposures produce non-
monotonic, tra nsgenerational r esponses in Daphnia magna. Environ. Pollut. 207,
176182.
Kiørboe, T., 1998. Population regulation and role of mesozooplankton in shaping marine
pelagic food webs. Hydrobiologia 363, 1327.
Kiørboe,T., 2007. Mate nding, mating, andpopulation dynamicsin a planktonic copepod
Oithona davisae: there are too few males. Limnol. Oceanogr. 52, 15111522.
Kiørboe, T., Ceballos, S., Thygesen, U.H., 2015. Interrelations between senescence, life-
history traits, and behavior in planktonic copepods. Ecology 96, 22252235.
Kiørboe, T., Mohlenberg, F., Hamburger, K., 1985. Bioenergetics of the planktonic copepod
Acartia tonsa: relation between feeding, egg production and respiration, and compo-
sition of specic dynamic action. Mar. Ecol. Prog. Ser. 26, 8597.
Kiørboe, T., Sabatini, M., 1995. Scaling of fecundity, growth and development in marine
planktonic copepods. Mar. Ecol. Prog. Ser. 120, 285298.
Klerks, P.L., Xie, L., Levinton, J.S., 2011. Quantitative genetics approaches to study evolu-
tionary processes in ecotoxicology; a perspective from research on the evolution of
resistance. Ecotoxicology 20, 513523.
Kwok, K.W.H., Souissi, S., Dur, G., Won, E.-J., Lee, J.-S., 2015. Copepods as References Spe-
cies in Estuarine and Marine Water. Academic Press, London, UK.
Medina, M., Barata, C., Telfer, R., Baird, D.J., 2002. Age- and sex-related variation in sensi-
tivity to thepyrethroid cypermethrin in the marine copepodAcartia tonsa Dana. Arch.
Environ. Contam. Toxicol. 42, 1722.
Moe, S.J., De Schamphelaere, K., Clements, W.H., Sorensen, M.T., Van den Brink, P.J., Liess,
M., 2013. Combined and interactive effects of global climate change and toxicants on
populations and communities. Environ. Toxicol. Chem. 32, 4961.
Morgan, A.J., Kil le, P., Sturzenbau m, S.R., 2007. Micr oevolution and ecotoxicology of
metals in invertebrates. Environ. Sci. Technol. 41, 10851096.
NationalResearch Council,2003. Oil in the Sea III:Inputs, Fates and Effects. National Acad-
emy Press, Washington, DC.
Nicolas, J.M., 1999. Vitellogenesis in sh and the effects of polycyclic aromatic hydrocar-
bon contaminants. Aquat. Toxicol. 45, 7790.
Nørregaard, R.D., Gustavson, K., Moller, E.F., Strand, J., Tairova, Z., Mosbech, A., 2015. Eco-
toxicological investigation of the effect of accumulation of PAH and possibleimpact of
dispersant in resting high arctic copepod Calanus hyperboreus. Aquat. Toxicol. 167,
111.
Nørregaard, R.D., Nielsen, T.G., Moller, E.F., Strand, J., Espersen, L., Mohl, M., 2014. Evalu-
ating pyrene toxicity on Arctic key copepod species Calanus hyperboreus. Ecotoxicol-
ogy 23, 163174.
Paffenhöfer, G.A., Stearns, D.E., 1988. Why is Acartia tonsa (Copepoda, Calanoida) restrict-
ed to nearshore environments. Mar. Ecol. Prog. Ser. 42, 3338.
Paine, R.T., Ruesink, J.L., Sun, A., Soulanille, E.L., Wonham, M.J., Harley, C.D.G., et al., 1996.
Trouble on oiled waters: lessons from the Exxon Valdez oil spill. Annu. Rev.Ecol. Syst.
27, 197235.
Pastorinho, R., Vieira, L., Re, P., Pereira, M., Bacelar-Nicolau, P., Morgado, F., et al., 2003.
Distribution, production, histology and histochemistry in Acartia tonsa (Copepoda:
Calanoida) as means for life history determination in a temperate estuary (Mondego
estuary, Portugal). Acta Oecol. 24, S259S273.
Pelletier, M.C., Burgess, R.M., Ho,K.T., Kuhn, A., McKinney, R.A., Ryba, S.A., 1997. Phototox-
icity of individual polycyclic aromatic hydrocarbons and petroleum to marine inver-
tebrate larvae and juveniles. Environ. Toxicol. Chem. 16, 21902199.
Perrichon, P., Akcha, F., Le Menach, K., Goubeau, M., Budzinski, H., Cousin, X., et al., 2015.
Parental trophic exposure to three aromatic fractions of polycyclic aromatic hydro-
carbons in the zebrash: consequences for the of fspring. Sci. Total En viron. 524,
5262.
Peterson,C.H., Rice, S.D., Short, J.W., Esler, D., Bodkin, J.L., Ballachey, B.E.,et al., 2003. Long-
term ecosystem response to the Exxon Valdez oil spill. Science 302, 20822086.
Prud'homme, S. M., Chaumot, A., Cassar, E., David, J.-P. , Reynaud, S., 2017. Impact of
micropollutants on the life-history traits of the mosquito Aedes aegypti: on the rele-
vance of transgenerational studies. Environ. Pollut. 220 (Part A), 242254.
Raisuddin, S., Kwo k, K.W.H., Leung, K.M.Y., Schlenk, D., Lee, J.S., 2007. The copepod
Tigriopus: a promising marine model organism for ecotoxicology and environmental
genomics. Aquat. Toxicol. 83, 161173.
Reddy, C.M., Arey, J.S., Seewald, J.S., Sylva, S.P., Lemkau, K.L., Nelson, R.K., et al., 2012. Com-
position and fate of gas and oil released to the water column during the Deepwater
Horizon oil spill. Proc. Natl. Acad. Sci. U. S. A. 109, 2022920234.
Reddy, C.M., Quinn, J.G., 2001. The North Cape oil spill: hydrocarbons in Rhode Island
coastal waters and Point Judith Pond. Mar. Environ. Res. 52, 445461.
Robertson, M., Schrey, A., Shayter, A., Moss, C.J., Richards, C., 2017. Genetic and epigenetic
variation in Spartina alterniora following the Deepwater Horizon oil spill. Evol. Appl.
http://dx.doi.org/10.1111/eva.12482.
Romero-Lopez, J., Lopez-Rodas,V., Costas, E., 2012.Estimating the capability of microalgae
to physiological acclimatization and genetic adaptation to petroleum and diesel oil
contamination. Aquat. Toxicol. 124, 227237.
Ross, K., Cooper, N., Bidwell, J.R., Elder, J., 2002. Genetic diversity and metal tolerance of
two marine species: a comparison between populations from contaminated and ref-
erence sites. Mar. Pollut. Bull. 44, 671679.
Samanta,S.K., Singh, O.V., Jain,R.K., 2002. Polycyclicaromatic hydrocarbons: environmen-
tal pollution and bioremediation. Trends Biotechnol. 20, 243248.
Sichlau, M.H., Hansen, J.L.S., Andersen, T.J., Hansen, B.W., 2011. Distribution and mortality
of diapause eggs from calanoid copepods in relation to sedimentation regimes. Mar.
Biol. 158, 665676.
Sichlau, M.H., Kiørboe, T., 2011. Age- and size-dependent mating performance and fertil-
ity in a pelagic copepod, Temora longicornis. Mar. Ecol. Prog. Ser. 442, 123132.
Støttrup,J.G., Richardson, K., Kirkegaard, E.,Pihl, N.J., 1986. The cultivation of Acartia tonsa
Dana for use as a live food source for marine sh larvae. Aquaculture 52, 8796.
Tang, K.W.,Dam, H.G., 2001. Phytoplankton inhibition of copepod egg hatching:test of an
exudate hypothesis. Mar. Ecol. Prog. Ser. 209, 197202.
Thor, P., Dupont, S., 2015. Transgenerational effects alleviate severe fecundity loss during
ocean acidication in a u biquitous plankt onic copepod. Glob. Chang. Biol. 21,
22612271.
Tiselius, P., Hansen, B., Jonsson, P., Kiorboe, T., Nielsen, T.G., Piontkovski, S., et al., 1995.
Can we use laboratory-rearedcopepods for experiments: a comparison of feeding be-
haviour and reproduction between a eld and laboratory population ofAcartia tonsa.
ICES J. Mar. Sci. 52, 369376.
Warton, D.I., Hui, F.K.C., 2011. The arcsine is asinine: the analysis of proportions in ecolo-
gy. Ecology 92, 310.
Wolska, L., Mechlinska, A., Rogowska, J., Namiesnik, J., 2012. Sources and fate of PAHs and
PCBs in the marine environment. Crit. Rev. Environ. Sci. Technol. 42, 11721189.
94 K.E. Krause et al. / Science of the Total Environment 607608 (2017) 8794
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    Microplastic (MP) leads to widespread pollution in the marine ecosystem. In addition to the physical hazard posed by ingestion of microplastic particles, concern is also on their potential as vector for transport of hydrophobic contaminants. We studied experimentally the single and interactive effects of microplastic and pyrene, a polycyclic aromatic hydrocarbon, on the swimming behaviour and predatory performance of juvenile barramundi (Lates calcarifer). Juveniles (18+ days post-hatch) were exposed to MPs, or pyrene (100 nM), or combination of both and feeding rate and foraging activity (swimming) were analysed. Exposure to MPs alone did not significantly influence feeding performance of the juveniles, while a concentration-response series of pyrene showed strong effect on fish behaviour when concentrations were above 100 nM. In the test of combined MP and pyrene exposure we observed no effect on feeding while swimming speed showed a significant decrease. Thus, our results confirm that short-time exposure to pyrene impacts performance of fish juveniles, while additional exposure to microplastic influenced their activity but not their feeding rate at the given conditions. Further studies on microplastics and other pollutants outlining their combined effects on behaviour and survival of tropical fish are encouraged.
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    To determine the optimal food concentrations for the mass culture of the tropical copepod Pseudodiaptomus annandalei at relevant temperatures, we conducted three functional response experiments. In these experiments, we quantified the grazing rate via the faecal pellet production of adult males and females. They were fed for 6 hr on one of three commonly used microalgal species in aquaculture: Chaetoceros muelleri, Isochrysis galbana and Tetraselmis chui at concentrations of 12.5–3,200 μg C L⁻¹ and three temperatures 25, 30 and 35°C. The number of pellets (PP) and the total volume of pellets (SPP) of both sexes increased rapidly with the increase in the microalgal concentrations until maximal pellet production (PPmax or SPPmax) was obtained, where females showed a consistently higher SPP than males. This pattern was similar for all three microalgal species. Males showed inconsistent PP and SPP in response to algae and temperatures. For females, they showed two clear patterns: a higher PP and SPP with increasing temperature from 25 to 30°C, then a lower PP and SPP at 35°C. Our study provides fundamental knowledge of pellet production to determine the food requirements of P. annandalei under different temperatures that are essential for designing the technical protocol for biomass culture.
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    Climate change and human activities induce an increased frequency and intensity of cyanobacterial blooms which could release toxins to aquatic ecosystems. Zooplankton communities belong to the first affected organisms, but in tropical freshwater ecosystems, this issue has yet been poorly investigated. We tested two questions (i) if the tropical Daphnia lumholtzi is capable to develop tolerance to an ecologically relevant concentration of purified microcystin-LR and microcystins from cyanobacterial extract transferable to F1 and F2 generations? And (ii) would F1 and F2 generations recover if reared in toxin-free medium? To answer these questions, we conducted two full factorial mutigenerational experiments, in which D. lumholtzi was exposed to MC-LR and cyanobacterial extract at the concentration of 1 μg L−1 microcystin continuously for three generations. After each generation, each treatment was spit into two: one reared in the control (toxin free) while the other continued in the respective exposure. Fitness-related traits including survival, maturity age, body length, and fecundity of each D. lumholtzi generation were quantified. Though there were only some weak negative effects of the toxins on the first generation (F0), we found strong direct, accumulated and carried-over impacts of the toxins on life history traits of D. lumholtzi on the F1 and F2, including reductions of survival, and reproduction. The maturity age and body length showed some inconsistent patterns between generations and need further investigations. The survival, maturity age (for extract), and body length (for MC-LR) were only recovered when offspring from toxin exposed mothers were raised in clean medium for two generations. Chronic exposure to long lasting blooms, even at low density, evidently reduces survival of D. lumholtzi in tropical lakes and reservoirs with ecological consequences.
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    Shallow, tropical marine ecosystems provide essential ecosystem goods and services, but it is unknown how these ecosystems will respond to the increased exposure to the temperature extremes that are likely to become more common as climate change progresses. To address this issue, we tracked the fitness and productivity of a key zooplankton species, the copepod Pseudodiaptomus annandalei, acclimated at two temperatures (30 and 34 °C) over three generations. 30 °C is the mean temperature in the shallow water of the coastal regions in Southeast Asia, while 34 °C simulated a temperature extreme that occurs frequently during the summer period. For each generation, we measured the size at maturity and reproductive success of individuals. In all three generations, we found strong negative effects of warming on all measured fitness-related parameters, including prolonged development time, reduced size at maturity, smaller clutch sizes, lower hatching success, and reduced naupliar production. Our results suggest that P. annandalei are already exposed to temperatures that exceed their upper thermal optimum. Increased exposure to extreme temperatures may reduce the abundance of these tropical marine copepods, and thus reduce the availability of resources to higher trophic levels. (Note: Accepted paper, details will be updated soon).
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    Within a given ecosystem, species persistence is driven by responses to the effects of biotic and abiotic stressors. Ongoing climatic shifts and increased pollution pressure have created the need to assess potential effects and interactions of physical and biotic factors on coastal ecosystem processes to project ecosystem resilience and persistence. In coastal marine environments, primary production dynamics are driven by the interaction between bottom-up abiotic effects and biotic effects induced by top-down trophic control. Given the many environmental and climatic changes observed throughout coastal regions, we assessed the effects of interactions among temperature, nutrients and grazing in a laboratory-based microcosm experiment. We did this by comparing chlorophyll-a (chl-a) concentrations at two temperatures in combination with four nutrient regimes. To test for and subsequent cascading effects on higher trophic levels, we also measured grazing and growth rates of the calanoid copepod Pseudodiaptomus hessei. We observed different phytoplankton and zooplankton community responses to temperature (17 °C, 24 °C) and nutrients (nitrogen only (N), phosphates only (P), nitrogen and phosphates combined (NP), no nutrient additions (C)). Contributions of predictors to model fit in the boosted regression trees model were phosphates (42.7%), copepods (23.8%), nitrates (17.5%) and temperature (15.9%), suggesting phosphates were an important driver for high chl-a concentrations observed. There was an increase in total phytoplankton biomass across both temperatures, while nutrient addition affected the phytoplankton community structure prior to grazing irrespective of temperature. Phytoplankton biomass was highest in the NP treatment followed by the N treatment. However, the phytoplankton size structure differed between temperatures, with microphytoplankton being dominant at 24 °C, while nanophytoplankton dominated at 17 °C. The (P and C treatments exhibited the lowest phytoplankton biomass. Copepod abundances and growth rates were higher at 17 °C than at 24 °C. This study highlights that bottom-up positive effects in one trophic level do not always positively cascade into another trophic level. It was, however, evident that temperature was a limiting factor for plankton abundance, productivity and community structure only when nutrients were limiting, with top-down pressure exhibiting minimal effects on the phytoplankton communities.
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    Biological invasions are largely considered to be a “numbers game”, wherein the larger the introduction effort, the greater the probability that an introduced population will become established. However, conditions during transport – an early stage of the invasion – can be particularly harsh, thereby greatly reducing the size of a population available to establish in a new region. Some successful non‐indigenous species are more tolerant of environmental and anthropogenic stressors than related native species, possibly stemming from selection (ie survival of only pre‐adapted individuals for particular environmental conditions) during the invasion process. By reviewing current literature concerning population genetics and consequences of selection on population fitness, we propose that selection acting on transported populations can facilitate local adaptation, which may result in a greater likelihood of invasion than predicted by propagule pressure alone. Specifically, we suggest that detailed surveys should be conducted to determine interactions between molecular mechanisms and demographic factors, given that current management strategies may underestimate invasion risk.
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    Background: Copepods make up the largest zooplankton biomass in coastal areas and estuaries and are pivotal for the normal development of fish larva of countless species. During spring in neritic boreal waters, the copepod pelagic biomass increases rapidly from near absence during winter. In the calanoid species Acartia tonsa, a small fraction of eggs are dormant regardless of external conditions and this has been hypothesized to be crucial for sediment egg banks and for the rapid biomass increase during spring. Other eggs can enter a state of induced arrest called quiescence when external conditions are unfavourable. While temperature is known to be a pivotal factor in the transition from developing to resting eggs and back, the role of pH and free Oxygen in embryo development has not been systematically investigated. Results: Here, we show in a laboratory setting that hypoxic conditions are necessary for resting eggs to maintain a near-intact rate of survival after several months of induced resting. We further investigate the influence of pH that is realistic for natural sediments on the viability of resting eggs and document the effect that eggs have on the pH of the surrounding environment. We find that resting eggs acidify their immediate surroundings and are able to survive in a wide range of pH. Conclusions: This is the first study to demonstrate the importance of hypoxia on the survival capabilities of A. tonsa resting eggs in a controlled laboratory setting, and the first to show that the large majority of quiescent eggs are able to tolerate prolonged resting. These findings have large implications for the understanding of the recruitment of copepods from sediment egg banks, which are considered the primary contributor of nauplii seeded to pelagic populations in nearshore habitats in late spring.
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    Copepods are excellent live feed for marine fish larvae in aquaculture. Culturing copepods at high density is important to increase the total egg yield, but this is still a main challenge. To address this, we conducted experiments to test factors affecting the egg harvest potential of the well studied and aquaculture relevant calanoid Acartia tonsa. A simple model was developed to evaluate the influence of individual egg production, egg predation, crowding effects and tank design on the egg harvest. At high densities from 500 to 3500 ind L−1, there was no difference in food ingestion and egg cannibalism. However, the copepods showed lower food consumption and egg cannibalism compared to the ecologically relevant densities of 20–100 ind L−1. Model calculations demonstrate that maximum egg harvest is the result of a subtle balance between water mixing and tank depth: a shallow, non-mixed tank will allow the eggs to settle and escape cannibalism but at the same time prevent the algal food staying suspended, and full utilization of the egg production potential depends on the fine tuning of these parameters.
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    Catastrophic events offer unique opportunities to study rapid population response to stress in natural settings. In concert with genetic variation, epigenetic mechanisms like DNA methylation may offer a mechanism of rapid response to organisms facing severe environmental challenges, and contribute to the high resilience of species like Spartina alterniflora, a foundation salt marsh grass which shows resilience to strong environmental disturbance. In 2010, the Deepwater Horizon oil spill devastated large portions of the coastline along the Gulf of Mexico. Following the spill, we simultaneously examined the genetic and epigenetic structure of recovering populations of S. alterniflora to oil exposure. We quantified genetic and DNA methylation variation using AFLP and MS-AFLP to test the hypothesis that response to oil exposure in S. alterniflora resulted in genetically and epigenetically based population differentiation. We found high genetic and epigenetic variation within and among sites, and found significant genetic differentiation between contaminated and uncontaminated sites, which may reflect non-random mortality in response to oil exposure. Additionally, despite a lack of genome wide patterns in DNA methylation between contaminated and uncontaminated sites, we found five MS-AFLP loci (12% of polymorphic MS-AFLP loci) that were correlated with oil exposure. Overall, our findings support genetically based differentiation correlated to exposure to the oil spill in this system, but also suggest a potential role for epigenetic mechanisms in population differentiation. This article is protected by copyright. All rights reserved.
  • Article
    Hazard assessment of chemical contaminants often relies on short term or partial life-cycle ecotoxicological tests, while the impact of low dose throughout the entire life cycle of species across multiple generations has been neglected. This study aimed at identifying the individual and population-level consequences of chronic water contamination by environmental concentrations of three organic micropollutants, ibuprofen, bisphenol A and benzo[. a]pyrene, on . Aedes aegypti mosquito populations in experimental conditions. Life-history assays spanning the full life-cycle of exposed individuals and their progeny associated with population dynamics modelling evidenced life-history traits alterations in unexposed progenies of individuals chronically exposed to 1 μg/L ibuprofen or 0.6 μg/L benzo[. a]pyrene. The progeny of individuals exposed to ibuprofen showed an accelerated development while the progeny of individuals exposed to benzo[. a]pyrene showed a developmental acceleration associated with an increase in mortality rate during development. These life-history changes due to pollutants exposure resulted in relatively shallow increase of . Ae. aegypti asymptotic population growth rate. Multigenerational exposure for six generations revealed an evolution of population response to ibuprofen and benzo[. a]pyrene across generations, leading to a loss of previously identified transgenerational effects and to the emergence of a tolerance to the bioinsecticide . Bacillus turingiensis israelensis (Bti). This study shed light on the short and long term impact of environmentally relevant doses of ibuprofen and benzo[. a]pyrene on . Ae. aegypti life-history traits and insecticide tolerance, raising unprecedented perspectives about the influence of surface water pollution on vector-control strategies. Overall, our approach highlights the importance of considering the entire life cycle of organisms, and the necessity to assess the transgenerational effects of pollutants in ecotoxicological studies for ecological risk assessment. Finally, this multi-generational study gives new insight about the influence of surface water pollution on microevolutionary processes.
  • Article
    Acartia tonsa is a calanoid copepod with high potential as live feed for marine aquaculture. However, its usage remains limited at an industrial scale, with cost effective production being conditional on successful culture at high density. The present study took an integrated approach to provide further insight on the effects of A. tonsa stocking density on copepod growth and adult reproduction, specifically egg production and egg hatching success. The effect of stocking density was studied by following the growth and survival of A. tonsa copepods, from egg hatching to maturity, on cultures initially stocked with 250, 400, 1000, 3000 and 6000 copepods l⁻¹. Additionally, the effects of high-density rearing, of adults kept at 100, 250, 500 and 2500 copepods l⁻¹, on egg production and hatching success were also evaluated over a 5-day period.
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    Following a brief overview of the patterns of ontogeny of osmoregulation in postembryonic stages, this review concentrates on the ontogeny of osmoregulation during the embryonic development of crustaceans, particularly in those species living under variable or extreme salinity conditions and whose hatchlings osmoregulate at hatch. Two situations are considered, internal development of the embryos in closed incubating, brood or marsupial pouches, and external development in eggs exposed to the external medium. In both cases, embryos are osmoprotected from the external salinity level and variation, either by the female pouches or by the egg envelopes. The mechanisms of osmoprotection are discussed. During embryonic life, temporary or definitive osmoregulatory organs develop, with ion transporting cells and enzymes such as Na+-K+ ATPase, permitting the embryos and then the hatchlings to osmoregulate and tolerate the external salinity.
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    On 20 April 2010, an explosion on the Deepwater Horizon drilling unit initiated an uncontrolled release of oil and gas from the Macondo seafloor well into the Gulf of Mexico that lasted for 87 days. Documenting and tracking the ecological, environmental, and human impacts of the Deepwater Horizon oil-well blowout has proved a considerable challenge. Nonetheless, valuable lessons continue to be learned, and data are revealing broad and substantial impacts on the Gulf ecosystem across a range of scales.
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    The optimal allocation of resources to repair versus reproduction in an organism may depend on the magnitude and pattern of the external mortality it is experiencing, which, in turn, may depend on its feeding and mate-finding behaviour. Thus, the fundamental activities of an organism, i.e., to feed, to survive, and to reproduce, are interrelated through trade-offs. Here, we use small planktonic copepods to examine how adult longevity and ageing patterns in a protected laboratory environment relate to the feeding mode (active searching vs. passive ambush feeding), mate-finding behaviour, and spawning mode of the species. We show that the average adult longevity varies between species by an order of magnitude and is independent of body size. Ambush feeders that carry their eggs have longer average life spans and experience high mortality later in life relative to active feeders that broadcast their eggs. Males generally have shorter life spans and experience high mortality earlier in life than females, and this difference may be accentuated in species where dangerous mate-finding is male biased. We finally show a trade- off between longevity and fecundity, with ambush feeders producing eggs at a rate 5-10 times lower than the active feeders, consistent with predictions from optimal resource allocation theory.
  • Article
    The optimal allocation of resources to repair vs. reproduction in an organism may depend on the magnitude and pattern of the external mortality it is experiencing, which, in turn, may depend on its feeding and mate-finding behavior. Thus, the fundamental activities of an organism, i.e., to feed, to survive, and to reproduce, are interrelated through trade-offs. Here, we use small planktonic copepods to examine how adult longevity and ageing patterns in a protected laboratory environment relate to the feeding mode (active searching vs. passive ambush feeding), mate-finding behavior, and spawning mode of the species. We show that average adult longevity varies between species by an order of magnitude and is independent of body size. Ambush feeders that carry their eggs have longer average life spans and experience higher mortality later in life relative to active feeders that broadcast their eggs. Males generally have shorter life spans and experience higher mortality earlier in life than females, and this difference may be accentuated in species where dangerous mate-finding is male biased. We finally show a trade-off between longevity and fecundity, with ambush feeders producing eggs at a rate five to 10 times lower than the active feeders, consistent with predictions from optimal resource allocation theory.