Lethal and sublethal effects of naphthalene and 1,2-dimethylnaphthalene on naupliar and adult stages of the marine cyclopoid copepod Oithona davisae

Lethal and sublethal effects of naphthalene and 1,2-dimethylnaphthalene on naupliar and adult stages of the marine cyclopoid copepod Oithona davisae

Environmental Pollution 157 (2009) 1219–1226 Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/lo...

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Environmental Pollution 157 (2009) 1219–1226

Contents lists available at ScienceDirect

Environmental Pollution journal homepage: www.elsevier.com/locate/envpol

Lethal and sublethal effects of naphthalene and 1,2-dimethylnaphthalene on naupliar and adult stages of the marine cyclopoid copepod Oithona davisae Enric Saiz a, *, Juancho Movilla a, Lidia Yebra a, Carlos Barata b, Albert Calbet a a b

Institut de Cie`ncies del Mar (ICM), CSIC, Ps. Marı´tim de la Barceloneta 37–49, E-08003 Barcelona, Catalunya, Spain ` stic Ambiental i Estudis de l’Aigua (IDAEA), CSIC, C/Jordi Girona 18–26, E-08034 Barcelona, Catalunya, Spain Departament de Quı´mica Ambiental, Institut de Diagno

The feeding activity of copepods is very sensitive to the direct and indirect (prey-viability mediated) effects of polycyclic aromatic hydrocarbons.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 April 2008 Received in revised form 23 September 2008 Accepted 3 December 2008

Short-term (24 h) exposure experiments have been conducted to determine the effects of two environmental relevant polycyclic aromatic hydrocarbons (PAHs), naphthalene (NAPH) and dimethylnaphthalene (C2-NAPH), on the naupliar and adult stages of the marine cyclopoid copepod Oithona davisae. To resemble more realistic conditions, those exposure experiments were conducted under the presence of food. The naupliar stages evidenced lower tolerance to PAH exposure regarding narcotic and lethal effects than adults. Copepod feeding activity showed to be very sensitive to the presence of the studied PAHs, detrimental effects occurring at toxic concentrations ca. 2–3 fold lower than for narcotic effects. In addition we report PAH-mediated changes in cell size and growth rate of the prey item, the heterotrophic dinoflagellate Oxyrrhis marina, that could indirectly affect copepod feeding and help explain hormesislike responses in our feeding experiments. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Zooplankton Larvae Polycyclic aromatic hydrocarbons Feeding Narcosis

1. Introduction Copepods are crucial components in marine planktonic food webs for their function as the main transfer node between primary producers and fish, and for their role in nutrient recycling and export (Miller, 2004). In spite of their high relevance in the marine ecosystem dynamics, the number of studies that address the potential impact of anthropogenic originated pollutants in marine planktonic copepods is still scarce and has concentrated mainly in acute lethal responses (Sverdrup et al., 2002). In this regard, the determination of toxic effects on the feeding activity of copepods has proved to be a very sensitive tool for assessing sublethal effects at the individual level (Barata et al., 2002a; Calbet et al., 2007). Short-term effects on feeding result into changes in growth and reproduction that will translate into the population level as lower recruitment and secondary production (Hutchinson et al., 1999; Barata et al., 2002a,b; Medina et al., 2002; Breitholtz et al., 2003; Wollenberger et al., 2003; Chandler et al., 2004; Kusk and Wollenberger, 2007; Turesson et al., 2007). Although other ecotoxicological studies on copepods have used fecundity as the target variable (i.e. Hutchinson et al., 1999; Barata et al., 2002a,b; Medina * Corresponding author. Tel.: þ34 93 230 9521; fax: þ34 93 230 9555. E-mail address: [email protected] (E. Saiz). 0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.12.011

et al., 2002; Breitholtz et al., 2003; Wollenberger et al., 2003; Chandler et al., 2004; Bellas and Thor, 2007; Kusk and Wollenberger, 2007; Turesson et al., 2007), significant effects on feeding are detectable at lower toxic concentrations, as evidenced by Barata et al. (2002a) and Calbet et al. (2007) for respectively, Tisbe battagliai and Paracartia (Acartia) grani. Furthermore, feeding responses have the advantage that can be determined also in non-reproducing developmental stages such as juveniles. Copepods are crustaceans that undergo one metamorphosis from the naupliar stage to the copepodite one, and six moltings on each of these two categories. Most ecotoxicological studies conducted on marine planktonic copepods studying sublethal effects on feeding or/and reproduction have dealt only with the adult stages (Barata et al., 2002a,b; Bellas and Thor, 2007; Kusk and Wollenberger, 2007; Turesson et al., 2007); studies on the earlier stages have mostly focused on the effects on survival and development (Hutchinson et al., 1999; Barata et al., 2002a; Medina et al., 2002; Breitholtz et al., 2003; Wollenberger et al., 2003; Chandler et al., 2004; Kusk and Wollenberger, 2007; Turesson et al., 2007). The aim of this study was to assess whether juvenile stages of marine copepods were more sensitive than adults to two of the most common petrogenic polycyclic aromatic hydrocarbons (PAHs) in seawater. In order to accomplish that we determined the lethal and sublethal effects of naphthalene and dimethylnaphthalene on

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the naupliar and adult stages of the marine planktonic copepod Oithona davisae. Besides the lower tolerance of juveniles to lethal and narcotic effects, we have observed a significant reduction on copepod feeding activity at sublethal concentrations of PAHs. In addition, we have ascertained the possibility of prey-mediated sublethal effects of PAHs on copepod feeding. The copepod Oithona davisae typically inhabits the coastal environment in the west Pacific, but nowadays, likely through ballast water, it is also found in the US west coast and in the Mediterranean (Ferrari and Orsi, 1984; Saiz et al., 2003). The genus of the marine planktonic cyclopoid copepod Oithona has been recently acquainted as very likely the most common copepod in the world oceans (Gallienne and Robins, 2001), inhabiting the coastal and the oceanic environments, and widely distributed from polar and temperate to tropical and equatorial ecosystems. It is surprising the lack of knowledge on the effects of environmentally relevant contaminants on this group of copepods, which often have a large contribution in terms of abundance and biomass in zooplankton communities. To our knowledge toxicological studies with this cosmopolitan and important genus of copepods are restricted to (Mironov, 1969, quoted in Corner, 1978) who reported lethal effects of whole oil on Oithona nana from the Black Sea, and to Barata et al. (2005), who addressed lethal and narcotic effects of several PAHs on adult stages of Oithona davisae. PAHs are important pollutants in the aquatic environment, originated from multiple input sources such as oil spills, tank cleaning, combustion of fossil fuels and atmospherically mediated transport from industrial sources. PAHs make up the most persistent toxicity of petroleum hydrocarbons after oil spills, because lighter components of the water soluble fraction, although toxic, are volatile and have shorter residence times in the aquatic environment (Neff et al., 1976; Albaige´s, 1989). Their toxicity increases with their lipophilicity, and they typically appear in mixtures, exhibiting an additive effect (Barata et al., 2005). The two PAHs studied here (naphthalene: NAPH, and dimethylnaphthalene: C2NAPH) are among the most abundant and toxic dissolved hydrocarbons in oil contaminated waters (Corner, 1978; Albaige´s, 1989; Pastor et al., 2001). 2. Methods 2.1. Experimental organisms The experiments were conducted with the naupliar (mostly stage 3, ca. 105 mm body length) and adult (ca. 300 mm prosome length) stages of the cyclopoid marine copepod Oithona davisae, reared under laboratory conditions for more than 100 generations (since year 2000). The copepods are routinely cultured in 10–15 l Perspex (GEHER) containers, at 19–20  C under a 12 h D:N light cycle and fed five times a week with a suspension of the heterotrophic dinoflagellate Oxyrrhis marina. The dinoflagellate is, at its turn, fed with a suspension of the cryptohyceae Rhodomonas salina, and kept under the same described conditions of light and temperature. A few days prior to collecting the specimens for the experiments, the Oithona davisae stock cultures were fed abundantly, to ensure the food supply was plentiful and to enhance egg production on females. For nauplii collection, 4 days before the experiment egg-bearing females from the stock culture were transferred into a hatching tank using a 130 mm sieve. After 15–20 h the adults were removed from the hatching tank, leaving the newly hatched nauplii cohort in the tank. Nauplii cohorts of Oithona davisae were allowed to grow 3 days on a diet of Oxyrrhis marina at a concentration of 1100 cell ml1 (ca. 3 ppm) before initiating the experiments, to ensure they have reached a fully feeding stage.

ULTRA Scientific (North Kingstown, RI, USA). Incubations were conducted at 20  C (12 h D:N light cycle) in 130 ml PyrexÒ glass bottles sealed with a Teflon screw lid, filled with a suspension of Oxyrrhis marina in 0.2 mm filtered seawater (ca. 450 and 350 cell ml1 for the experiments with NAPH and C2-NAPH, respectively) and the corresponding toxic concentration. For each toxic concentration there were six replicate bottles: two with no added copepods, serving as control reference for estimating copepod feeding rates; two with adult copepods; and two with nauplii. Groups of 30 adult females were individually picked out from the cultures and set aside in small vials for posterior transferring to experimental bottles. For nauplii, known concentrations of nauplii were prepared in a beaker and aliquots pipetted directly to the experimental bottles to achieve the desired concentration (ca. 200 individuals per bottle). Some extra samples of nauplii and adults were preserved for posterior body size measurements. After capping, the bottles were placed on a Ferris wheel and let rotate (0.2 rpm) through the incubation. After ca. 24 h the bottles were taken out and their content screened through a submerged 40 mm sieve to gently collect the copepods. For each bottle a 75-ml sample of the screened Oxyrrhis marina suspension was preserved in 1% acid Lugol’s solution for posterior counting and cell sizing. The collected copepods were immediately transferred to small Petri dishes filled with toxic-free 0.2 mm filtered seawater for monitoring swimming activity and survival; after 4–6 h the copepods were checked again for recovery after the removal from the toxic. Acetone (HPLC grade, <0.5 ml l1) was used as a carrier of the PAHs in all the experiments. Although previous studies with this and other species of copepods (Barata et al., 2005; Calbet et al., 2007) have shown that the low concentrations of acetone used have no effects on copepod survival and activity, to ensure comparison, the bottles with no added toxic in our experiments had the same amount of acetone. 2.3. Chemical analyses To account for possible effects of prey (Oxyrrhis marina) on water phase concentrations and hence on the exposure doses of the studied PAHs, an additional experiment was conducted to evaluate such potential losses. The experiment consisted of eight exposure treatments that included two concentrations of NAPH (1000, 7000 mg l1) and of C2-NAPH (200, 1000 mg l1) with and without the presence of food (Oxyrrhis marina at ca. 430 cells l1). The remaining experimental conditions were identical to the ones used in toxicity tests. Initial and final (after ca. 24 h exposure) PAH concentrations in the dissolved phase were analysed by GC-MS using a slight modification of the method described by Martinez et al. (2004). Briefly, 30 ml of each sample was filtered through glass fiber filters (Whatman GF/F, 25 mm diameter), spiked with the surrogate solution (100 ng acenaphtene-d10), preconcentrated in solid-phase C18 extraction cartridges (500 mg, Merck) using a Baker vacuum system, dried under vacuum and then eluted with 20 ml of hexane:dichloromethane (1:1, v/v). The extract was concentrated to 0.5 ml under a gentle stream of nitrogen, transferred to 1.7 ml amber vials and reconstituted with 400 ml of hexane. Naphthalene and dimethylnaphthalene were analyzed by gas chromatography coupled to a Trace mass spectrometer (ThemoQuest, Barcelona, Spain) and quantified with the internal standard procedure. The system was operated in electron impact mode (EI, 70 eV) and 1 ml of sample was injected in the splitless mode with the source and interface temperature set at 250  C. The separation was achieved with a 30 m  0.25 mm i.d.  0.25 mm HP5-MS capillary column (Agilent, Germany). The oven temperature was programmed from 70  C (holding time 1 min) to 250  C at 18  C/min (holding time 2 min). With these conditions, naphthalene eluted at 5.51 min and dimethylnaphthalene at 7.61 min and were quantified using the base peak at m/z 128 and 141, respectively (m/z 188 for the surrogate standard anthracene d10). The analytical procedure was validated by determining the recovery rates using ultrapure water. Recoveries were above 80% at a spiking level of 10 mg l1 and good reproducibility (between 4 and 6%) was found. Blank analysis revealed no contribution of any of the target compounds. Losses of PAH concentrations in the water phase after 24 h exposures were within 6 and 23% of initial values (Table 1). Measured mean PAH concentration during the incubations for NAPH were 82 and 79% of nominal concentrations for the treatment without and with food (Oxyrrhis marina), respectively; for C2-NAPH measured mean concentrations were 87 and 84% of nominal values, respectively. Accordingly, nominal concentrations in the experiments were converted into actual mean ones using the determined loss factors under food presence: 0.79 and 0.84 for NAPH and C2-NAPH, respectively.

Table 1 PAH dissolved concentrations (mg l1) in control and treatments at the start (t0) and after 24 h of exposure (t24).

2.2. Lethal and sublethal response experiments The experiments consisted of 24 h incubations of naupliar and female adult stages of Oithona davisae under the presence of food, the dinoflagellate Oxyrrhis marina, at a range of different concentrations of NAPH (nominal: 0, 125, 250, 500, 1000, 3000, 7000, 10,000 and 15,000 mg l1) and C2-NAPH (nominal: 0, 50, 100, 200, 300, 500, 1000, 2000 and 3000 mg l1). NAPH (>96% purity) was purchased from Sigma–Aldrich (St. Louis, MO, USA), and C2-NAPH (>97% purity) was obtained from

PAHs

Nominal

C2-NAPH

200 1000

t0 180  4 920  17

t24 without food 170  2 820  19

148  3 780  15

NAPH

1000 7000

905  36 6200  236

790  35 4900  206

700  35 4760  233

Average concentrations of two replicates (SD) are shown.

t24 with food

E. Saiz et al. / Environmental Pollution 157 (2009) 1219–1226 2.4. Calculations The carbon content of Oxyrrhis marina was obtained from cell volume by the conversion factor 0.123 pg C mm3 provided by Pelegrı´ et al. (1999) for this species. Cell volume of O. marina was estimated, assuming ellipsoidal shape, by measuring cell linear dimensions on digital pictures taken under the microscope using the ImageJ software (National Institute of Health, Bethesda, MD, USA). The body size (BS, mm) of Oithona davisae (adults: prosome length; nauplii: total length) was measured either directly under the stereomicroscope with an eyepiece micrometer (adults) or taking digital pictures under a microscope (nauplii) and using ImageJ software. Body size was converted into body mass (BM, mg C) using the equation provided by Uye and Sano (1998) for this copepod species: BM ¼ 1:83  106  BS2:05

(1)

Survival rate, as % of the incubated individuals that survived to the 24 h toxic exposure, was estimated from the number of dead (not swimming after gently sticking with a laboratory needle) individuals at the second visual checking. Narcosis, as % of survivors that did exhibit narcotic effects (evidenced as lack of motility) was estimated from the difference in the number of non-swimming individuals at the first checking (which included actual dead animals plus the narcotized ones) and the second checking (which included only those copepods that did not recover from toxic effects). Data for survival and narcosis were fitted to a slightly modified Hill allosteric decay regression model according to Barata et al. (2002a): y ¼

a bc bc þ ðlogðxÞÞc

(2)

where y is either the survival or narcosis response, x is the toxic concentration, a is either the maximum survival or narcosis, b is the respective logarithmic 50%

reduction concentrations, and c is the shape coefficient. For fitting purposes after logarithmic transformation, toxic concentration in the bottles with absence of toxic was adjusted to 1 mg l1, one to two orders of magnitude lower than the lowest toxic concentration tested. Median lethal concentration (LC50) and half maximal effective narcotic concentration (EC50) were calculated by detransforming log estimated b parameters. Clearance and ingestion rates of adults and nauplii were estimated from cell removal and calculated according to Frost (1972), after verification that prey growth rates in grazing bottles were significantly lower than in the control bottles (one-way ANOVA test, two-tailed P < 0.05). Aliquots of the grazing and control bottle samples were settled in 10 ml Utermo¨hl chambers and counted under an inverted microscope. In order to explore indirect effects of PAHs on copepod feeding mediated through changes on prey (Oxyrrhis marina) viability, we also evaluated the changes in O. marina cell size and growth rate in the control bottles (with no grazing) as a function of PAH concentration.

3. Results 3.1. Survival and narcotic effects Overall nauplii showed higher sensitivity to the studied PAH in both lethal and narcotic effects (Fig. 1). Regarding NAPH, adult Oithona davisae did not exhibit any significant mortality at the concentrations tested (10 mg l1), whereas for nauplii 24 h LC50 were 4422 (95% CI: 3942–4961) mg l1. NAPH also provoked significant narcotic effects on Oithona davisae; again nauplii were

Nauplii Oithona davisae NAPHTHALENE

Adult Oithona davisae NAPHTHALENE 100

% narcosis or survival

% narcosis or survival

100

75

50 survival narcosis 25

0

75

50

25

0 0.1

101

1

102

103

104

PAH concentration (µg

105

0.1

l-1)

1

101

102

103

PAH concentration (µg

Nauplii Oithona davisae DIMETHYLNAPHTHALENE

104

105

l-1)

Adult Oithona davisae DIMETHYLNAPHTHALENE

100

100

% narcosis or survival

% narcosis or survival

1221

75

50

25

0

75

50

25

0 0.1

1

10

1

2

10

3

10

PAH concentration (µg

l-1)

10

4

0.1

1

101

102

PAH concentration (µg

103

104

l-1)

Fig. 1. Survival and narcotizing responses of nauplii and adults of Oithona davisae to naphthalene and dimethylnaphthalene. Data have been fitted to an allosteric decay model, except for the survival response of adults to naphthalene, where no mortality response was observed. Each point represents a single measure.

E. Saiz et al. / Environmental Pollution 157 (2009) 1219–1226

quite more sensitive than adults, with EC50 for narcosis being 4276 (95% CI: 3791–4822) mg l1 and 6327 (95% CI: 6025–6645) mg l1 for nauplii and adults, respectively. LC50 and EC50 for nauplii manifest the drastic effects of NAPH on copepod nauplii. At NAPH concentrations ca. 4300–4400 mg l1, half the population was dead, and of the remaining live animals half were narcotized (and, likely, eventually dying if exposure prolonged and feeding constrained). C2-NAPH proved stronger toxic effects on Oithona davisae than the parental compound, with lethal and sublethal responses evidenced at concentrations six to eight times lower (Fig. 1). Both nauplii and adult copepods exhibited mortality under 24 h exposure to C2-NAPH with LC50 being respectively, 771 (95% CI: 759– 784) mg l1 and 1346 (95% CI: 1047–1732) mg l1. Narcotic effects occurred at slightly lower C2-NAPH concentrations than the respective LC50, EC50 for nauplii and adult copepods being 523 (95% CI: 156–1758) mg l1 and 983 (95% CI: 384–2517) mg l1, respectively. Fig. 2 shows the feeding rates of Oithona davisae under exposure to NAPH and C2-NAPH, expressed either as clearance rates (ml swept clear by the copepod) or as daily rations (daily intake as percentage of own body carbon). Although the feeding experiments were conducted concurrent to the survival incubations, samples were analyzed only for those concentrations without lethal or narcotic effects (NAPH <4000 mg l1; C2-NAPH <500 mg l1; Fig. 1). For both compounds the 24-h exposure resulted in a sharp decline of clearance rates at high PAH concentrations (ca. 2000 mg l1 for NAPH, and ca. 200–250 mg l1 for C2-NAPH). At intermediate PAH concentrations, however, the response was variable, often showing

adult nauplii 4

0.8

3

0.6

2

0.4

1

0.2 0 101

1

102

103

104

Copepods are known to be especially sensitive to xenobiotic toxins, and for this reason they have been proposed as a target group for ecological testing of environmental threats (Kusk and Petersen, 1997; Raisuddin et al., 2007). Here we have reported

Oithona davisae NAPHTHALENE 250 200 150 100 50 0 0.1

101

1

102

103

PAH concentration (µg l-1)

PAH concentration (µg l-1)

Oithona davisae DIMETHYLNAPHTHALENE

Oithona davisae DIMETHYLNAPHTHALENE

5

1

4

0.8

3

0.6

2

0.4

1

0.2

0 0.1

4. Discussion

1

101

0 103

102

PAH concentration (µg

l-1)

Daily ration (as % body carbon ingested daily)

0 0.1

Nauplius clearance rate (ml ind-1 d-1)

Adult clearance rate (ml ind-1 d-1)

1

5

Nauplius clearance rate (ml ind-1 d-1)

Adult clearance rate (ml ind-1 d-1)

Oithona davisae NAPHTHALENE

some enhancement of clearance rate responses (Fig. 2). In terms of daily rations (% carbon weight ingested daily), the patterns were similar. As a consequence, it was not possible to model accurately observed responses using the allosteric decay function (Eq. (2)). Nevertheless, Fig. 2 results suggest that nauplii had equivalent and lower sensitivities to NAPH and C2-NAPH than adults, respectively. NAPH and C2-NAPH also had an effect on the prey, Oxyrrhis marina, during the experiments of exposure of Oithona davisae to PAHs (Fig. 3). Both PAHs resulted in a decrease in the cell number of Oxyrrhis marina during the incubations as a function of PAH concentration. At the highest concentrations of C2-NAPH visual observation under the microscope during counting evidenced that many cells had swollen and some exploded (cell remains were found), making it unfeasible to obtain reliable counts and size determinations at those high PAH concentrations. Regarding changes in cell volume, for NAPH there is a decline in cell volume at higher concentrations (up to 40% reduction in cell volume with respect to no toxic exposure, Fig. 3). However, for C2-NAPH there were no clear patterns of cell volume change (likely due to the above-mentioned unfeasibility to determine cell size at high C2-NAPH concentrations).

Daily ration (as % body carbon ingested daily)

1222

104

250 200 150 100 50 0 0.1

1

101

102

PAH concentration (µg

103

l-1)

Fig. 2. Effects of PAHs on the feeding performance of Oithona davisae on the heterotrophic dinoflagellate Oxyrrhis marina. Results are depicted as mean  SE. Notice that on left graphs the scales for nauplii and adult clearance rates are different.

E. Saiz et al. / Environmental Pollution 157 (2009) 1219–1226

Oxyrrhis marina NAPHTHALENE

Oxyrrhis marina NAPHTHALENE 2500

Final cell volume (µm3 cell-1)

Final concentration in -1 control bottles (cells ml )

1000 800 600 400 200 0

0.1

1

101

102

103

104

1500 1000 500

1

101

102

103

104

PAH concentration (µg l-1)

PAH concentration (µg l-1)

Oxyrrhis marina DIMETHYLNAPHTHALENE

Oxyrrhis marina DIMETHYLNAPHTHALENE

105

2500

Final cell volume (µm3 cell-1)

800 600 400 200 n.d. 0 0.1

2000

0 0.1

105

1000

Final concentration in -1 control bottles (cells ml )

1223

2000 1500 1000 500 n.d. 0

1

101

102

103

PAH concentration (µg

104

l-1)

0.1

1

101

102

103

PAH concentration (µg

104

l-1)

Fig. 3. Cell concentration (end values, two replicates shown) and cell volume (geometric mean  SE) of the heterotrophic dinoflagellate Oxyrrhis marina under PAH exposure in control bottles. n.d.: not determined because many cells swelled and exploded (see text).

experimental data on the lethal and sublethal (narcosis, feeding) effects of two environmental relevant components of the water soluble fraction of fuel oil, the polycyclic aromatic hydrocarbons NAPH and C2-NAPH, on the naupliar and adult stages of the marine cyclopoid copepod Oithona davisae. Our short-term acute response experiments with PAHs are not easy to compare with previous studies with other planktonic copepods, firstly because often water-accommodated oil fractions have been used (e.g. Berdugo et al., 1977; Berman and Heinle, 1980; Hebert and Poulet, 1980–81; Bejarano et al., 2006) instead of single compounds, and secondly, because target species and incubation times varies among studies according to the scientist’s interests. In addition, standard protocols for acute response typically are based on 48 h incubations under starvation (International Organization for Standardization, 1999). Although water soluble hydrocarbons can be acquired by copepods directly from the aqueous phase through cutaneous absorption, it is known that dietary intake of phytoplankton, which can absorb PAH dissolved in the water, can be a major path of uptake (Corner, 1978; Wang and Wang, 2006). Hence, our acute experiments were conducted under the presence of food to resemble more realistic situations. For this reason one would expect our estimated LC50 values to be lower than those reported under starvation conditions (Barata et al., 2005). However, the comparison with the work of Barata et al. (2005), where the adult female stage of the same copepod species was studied under starving conditions in 48-h exposure to PAHs (their Table 2), evidences the opposite, starving adult copepods being about 2 fold

more sensitive (LC50 for NAPH and C2-NAPH were, respectively, 7000 and 600 mg l1) than the ones in our experiments. Very likely, 48-h starvation for small copepods like Oithona davisae, which do not store large amounts of lipid in their body, is sufficient to weaken the vigor of the organism and increase their sensitivity to toxics. For instance, Saiz et al. (1997) showed that the egg production rates of small copepods like the ones belonging to the genus Acartia, Centropages, Temora or Oithona, with small lipid reserves, responded drastically to changes in food availability within the 24 h. In our experiments, even if copepods increased their exposure to the studied contaminants by ingesting contaminated food items, at the same time were allowed, within some extent, to satisfy their metabolic requirements in comparison to the starving copepods in the study of Barata et al. (2005). Furthermore, Medina et al. (2002) and Barata et al. (1999) have also reported that the sensitivity of small planktonic crustaceans to toxic stress may increase over 2 fold from 24 to 48 h of exposure time. This probably also explains the puzzling result that under the range of NAPH concentrations tested in our work (<15,000 mg l1), this PAH did not induce any significant mortality on adult Oithona davisae whereas it did at around 7000 mg l1 in the study of Barata et al. (2005). Regarding sublethal effects, both PAHs produced clear narcosis on adult Oithona davisae, with EC50 being lower than the corresponding LC50. Other previous studies (Berdugo et al., 1977; Calbet et al., 2007), including that of Barata et al. (2005) on Oithona davisae, had already reported the presence of PAH-mediated narcotic effects on copepods and their reversible character after

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postexposure in toxic-clean (control) water (Berdugo et al., 1977; van Wezel and Opperhuizen, 1995). Surprisingly, narcotic effects were less evident on the naupliar stages. We think this difference is likely because narcotic effects may be the prelude of eventual lethal ones when exposure is prolonged. It seems reasonable that the naupliar stages could cope less well with the consequences of being narcotized (i.e. not feeding and ultimate death) than adults. This observation falls within the general trend of higher toxic sensitivity of the juvenile stages observed in copepods in studies dealing with other compounds (e.g. Pseudodiaptomus marinus vs. bis(tributyltin) oxide: Huang et al., 2006; Acartia tonsa vs. cypermethrin: Medina et al., 2002). The action of PAHs on invertebrates is typically mediated by non-polar narcosis, where the partition of lipophilic toxics on the cell membrane affects the bounded enzyme systems, e.g. those involved in ion regulation (Darville et al., 1983). The higher sensitivity to the methylated form observed in our study, both for lethal and sublethal effects, is in well agreement with the abovementioned mode of action and hence their toxicity is proportional to their octanol-water partition coefficient (Ott et al., 1978; Barata et al., 2005). However, although there are evidences of the accumulation and biotransformation of PAHs in copepods (Harris et al., 1977; Lotufo, 1998), it is less known their mode of action at the functional and physiological level. For instance, in vertebrates PAHs can induce abnormalities and malfunctions in fish embryos (e.g. Incardona et al., 2004). Recent studies at the molecular level on the calanoid copepod Calanus finmarchicus indicate that naphthalene toxicity does not induce a detoxification system mediated by the membrane-bound cythochrome P450 enzyme family as in some vertebrates, but results in lipid peroxidation, presumably affecting egg and sperm production (Hansen et al., 2008). In this regard Lotufo (1998) found in the harpacticoid copepod Coullana sp. that a significant fraction of the fluoranthene body residue was allocated to the lipid rich maturating eggs eventually leaving the body when the eggs were laid. Although the reduction in copepod fecundity observed after exposure to PAHs in several studies (Ott et al., 1978; Bellas and Thor, 2007; Calbet et al., 2007; Jensen et al., 2008) is more likely driven by a toxic-mediated reduction in feeding activity (Calbet et al., 2007), the above-mentioned finding could set a mechanism of action involved in the reduction of egg hatching success under exposure to PAHs, as reported by other authors (Jensen et al., 2008; Bellas and Thor, 2007). Regarding the effects of PAHs on the feeding rates of Oithona davisae, our study show that this variable is very sensitive to their presence, detrimental effects for feeding occurring at toxic concentrations ca. 2–3 fold lower than for narcotic effects. Probably, this lower threshold for feeding effects is a consequence of very subtle, preliminary drowsy effects disturbing feeding previous to the full narcosis at higher toxic concentrations that we observed (detectable as not moving organisms). Similar high sensitivity of feeding activity to toxic exposure has been previously observed in other calanoid copepods (Acartia tonsa vs. cypermethrin: Medina et al., 2002; Acartia grani vs. PAHs: Calbet et al., 2007; Calanus spp. vs. pyrene: Hjorth et al., 2007; Eurytemora affinis vs. petroleum hydrocarbons: Berdugo et al., 1977). It is a bit puzzling that the feeding activity of adult Oithona davisae appears more sensitive to C2-NAPH than that of the naupliar stages, whereas this stage-dependent difference is not so conspicuous in the case of exposure to NAPH. The variability in the data at intermediate toxic concentrations (see comments on hormetic-like response below), however, difficult to robust comparisons. There are arguments to think that the feeding activity of the adult stage is likely more sensitive than the naupliar ones. When feeding rates are expressed in weight-specific terms (daily ration), it is apparent that the intake of presumably toxic-loaded O. marina

is similar between the nauplii and the adults in the experiments conducted with C2-NAPH. In the experiments with exposure to NAPH, the detrimental effects occurred at similar toxic concentrations, although nauplii showed much higher weight-specific ingestion rates than adults. This unexpected higher sensitivity of adults could be explained by the fact that adults have much more developed and complex feeding appendages and motoneuronal systems, being more prone to the narcotic effects of PAHs. Supporting this hypothesis Berman and Heinle (1980) observed that low exposures to water-accommodated fuel oil modified significantly the feeding behavior of the adult and late copepodite stages of the copepod Acartia, changing their prey selectivity pattern. Additional studies are needed to further evidence such response in copepods and to confirm these hypotheses. Another interesting observation from our results is that in comparison with the controls without PAHs, as PAH concentration increases there appears to be an enhancement of feeding rates previous to the drastic decline. Although we cannot discard the presence of hormesis (Calabrese, 2005; Bejarano et al., 2006) or changes in feeding behavior such as those observed by Berman and Heinle (1980) to explain the enhancement of feeding, very likely in our experiments the observed changes in Oxyrrhis marina growth, viability and size would certainly affect indirectly the ingestion rates of zooplankton. For instance, it is well known copepod feeding rates are dependent on prey size (e.g. Berggreen et al., 1988). In addition, the decrease in cell number and the cell volume alteration indicate changes in Oxyrrhis marina physiology that could be reflected as well in variables such as swimming performance of the cell, nutritional content, etc., that can affect copepod feeding performance (e.g. Broglio et al., 2001; Henriksen et al., 2007). 5. Conclusions Testing toxic effects on copepods under starvation may severely bias the threshold toxic concentrations eliciting negative effects, because food intake may represent the major path for toxic incorporation in copepods, and also because of a weakening effect under starving for copepods with low lipid reserves. The juvenile (naupliar) stages of Oithona davisae appear more sensitive to acute exposures to PAHs than the adults. Significant detrimental effects of the studied hydrocarbons on the feeding of marine copepods seem to occur at concentrations much lower than those where full narcosis and death occurs. The environmental risk of Oithona davisae to PAH exposure, however, except for areas particularly polluted (e.g. vicinity of oil spills, tank cleaning), would appear to be low because natural concentrations of PAHs (1–100 mg l1; Barbier et al., 1973; Witt, 1995; Law et al., 1997; Doval et al., 2006) are far below the effective PAH concentrations resulting on detrimental effects on Oithona davisae observed in our study. Nevertheless, taking into account that PAHs typically occur in mixtures, exhibiting an additive effect (Barata et al., 2005), and that long time exposure was not tested in our experiments, detrimental effects on natural populations of Oithona may occur at much lower PAH concentrations. We have not contemplated in our study UV light enhancement of PAH toxicity (e.g. Bellas and Thor, 2007), a factor that could significantly diminish the toxic thresholds reported here. Typically the bulk of copepods remain in relatively deeper waters during daytime and the amount of UV radiation reaching such depths would be negligible. However, at night they migrate into less deep waters and there they could encounter photomodified products (depending on the penetration of UV light). For this reason it is not clear how relevant this UV-mediated effect could be for copepods under natural circumstances. Finally the observed changes in Oithona davisae prey in our experiments, the dinoflagellate Oxyrrhis marina, can explain some

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