Article pubs.acs.org/est
Sublethal Exposure to Crude Oil Enhances Positive Phototaxis in the Calanoid Copepod Calanus finmarchicus Cecilie Miljeteig,*,† Anders Johny Olsen,† Trond Nordtug,‡ Dag Altin,§ and Bjørn Munro Jenssen† †
Department of Biology, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway SINTEF Materials and Chemistry, Marine Environmental Technology, NO-7465 Trondheim, Norway § BioTrix, NO-7022 Trondheim, Norway ‡
S Supporting Information *
ABSTRACT: We investigated the effects of exposure to sublethal concentrations of the water accommodated fraction (WAF) of fresh crude oil on phototactic behavior of the calanoid copepod Calanus f inmarchicus (Gunnerus) copepodite stage 5 (C5). Exposure was conducted in closed bottle systems, and behavior was tested in a tailored setup. Exposure times were 24, 48, 72, and 96 h, and the chosen exposure concentration was 25% of the recorded LC50 value for the WAF (309 ± 32 μg/L total hydrocarbon, including 20.37 ± 0.51 μg/L total PAH). The exposure significantly increased the positive phototactic behavior of the copepods after 24 h exposure and a similar significant effect was observed for all exposure durations. Additionally, experiments were conducted with nonexposed copepods with low lipid reserves. The main effect of the exposure was a shift in the response to light toward a more positive phototaxis, similar to that observed in nonexposed C. f inmarchicus with low lipid reserves. The observed change in phototactic behavior observed in these studies suggests that the depth distribution of this species could be altered following an oil spill. Thus, further research is warranted to determine the possible interactive effects of light and oil spill exposures on Calanus population dynamics under field conditions.
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INTRODUCTION
Lipids have been reported to constitute up to 76% of dry mass in adult females and C5s.6 Many zooplankton, including C. f inmarchicus, perform diel vertical migration, with the most common pattern being to ascend to the surface water at dusk to feed at night and to migrate to greater depths at dawn and remain in the deeper water layers during daytime. The migration depth may vary from a few meters to hundreds of meters.7,8 The widely supported hypothesis for these migrations is the predator avoidance hypothesis, suggesting that the zooplankton reduce the risk of predation by visually hunting predators by avoiding the surface water layer during daytime.9,10 Light is generally considered the most important signal controlling these migrations, by inducing a phototactic response in the animals,11 although a wide range of other factors, such as food concentration, predator presence, temperature or hunger, may influence the magnitude or direction of the response.12,13 Phototaxis is defined as a directional movement relative to a directional light source, thus positive phototaxis is movement toward the light source and negative phototaxis is movement away from the light source.14 Lipid content or energy reserves
In marine ecosystems discharges of chemicals from petroleum activities are among the most important anthropogenic pollutant stressors. Marine discharges of petroleum components through operational discharges or larger accidents and leaks may be substantive and lead to increased exposure of marine organisms including epipelagic and mesopelagic plankton.1 The calanoid copepod Calanus f inmarchicus (Gunnerus) is considered an ecological key species in the North Atlantic pelagic ecosystem,2 and is the dominant copepod species in the northern part of the North Sea, the Norwegian Sea, and in the Atlantic inflow region to the Barents Sea. The species contributes to a very large fraction of the total zooplankton biomass (up to 90%) in these waters,3 and represents one of the most important transfer routes of energy and matter between the primary production of phytoplankton and fish species such as Atlantic cod Gadhus morhua L. and herring Clupea harengus L.4 C. f inmarchicus goes through six nauplii stages and five copepodite stages before reaching the adult stage, with overwintering (diapause) primarily occurring at copepodite stage 5 (C5).5 During the copepodite stages the species accumulate and store energy reserves as lipids in an internal elongated oil sac, and large amounts of lipids may be accumulated under favorable feeding conditions.5 The stored lipids are primarily used for gonad formation in the adult stage.6 © 2013 American Chemical Society
Received: Revised: Accepted: Published: 14426
August 24, 2013 November 8, 2013 November 12, 2013 November 12, 2013 dx.doi.org/10.1021/es4037447 | Environ. Sci. Technol. 2013, 47, 14426−14433
Environmental Science & Technology
Article
used for preparing the exposure solutions. To reduce evaporation from the system, the bottle was closed during stirring. The exposure concentration was determined from previous stage-dependent LC50 tests with the species.23 An exposure concentration corresponding to 25% of the median lethal concentration after 96 h exposure of C5 was chosen, as no mortality was observed at this concentration.23 The LC50 for the fresh oil used corresponds to 64% of the undiluted WAF, and the dilution used for the current experiment was therefore 16% of the original WAF, corresponding to an average concentration of 310 μg total petroleum hydrocarbon (TPH) including carbon numbers from 5 to 36 (C5−C36) in the exposure media. The exposure system consisted of 2 L bottles filled with WAF diluted with filtered seawater (1 μm cartridge filter, CUNO, CT, USA) to obtain the desired concentration. A total of 60 C5 C. finmarchicus were added to each bottle, and the bottles were capped with Teflon-lined screw caps leaving virtually no head space. For each exposure bottle a paired control filled with filtered natural seawater and 60 C5 C. f inmarchicus was prepared. The copepods were not fed during exposure, and were kept in the same light regime as the cultures (18:6 h of light/dark) during the entire exposure period. The durations of the exposures were 24, 48, 72, and 96 h under static exposure. Exposure Experiments. Experiments investigating phototactic behavior were conducted in a conditioning room with air and water temperatures of 10 (±1) °C in complete darkness, except for the light stimuli and near-infrared light for video recording. A total of 6 replicates of exposure bottles and 6 replicates of control bottles (each containing well-fed 60 copepods that had large oil sacs and thus adequate lipid reserves) were prepared for each exposure series (24, 48, 72, and 96 h). For each time series, the cohort in each of the control and exposure bottles were tested separately, with three controls and three exposure groups tested per day, alternating control and exposed groups. To obtain a total of 6 replicates for each exposure series (24 to 96 h), the WAF generation and the subsequent exposure and behavioral experiments were conducted in two rounds. For the low-lipid groups (lean control), the same number of replicates (6 per exposure time) was subsequently prepared with C5 C. f inmarchicus that were less well-fed and thus had significantly lower lipid reserves (t test: t = 39.1, P < 0.001). Experimental Setup for Testing Phototaxis. The experimental setup for investigating phototactic behavior included an aquarium with a light stimulus at one end of a raceway.24 The aquarium had the dimensions 50 cm × 50 cm × 12 cm and was made of 10 mm glass (Pilkington Optiwhite, NSG Co., Ltd., Japan). Glass walls (8 mm, Pilkington Optiwhite) constructed a raceway inside the aquarium limiting the projection area available to the copepods to 50 cm × 13 cm. All experiments were conducted with a water depth of 8 cm. The light stimulus source was a white light emitting diode (LED; Luxeon I Lambertian, 350 mA, Phillips Lumileds, LXHL-PW01). Absorptive neutral density filters (CVI Melles Griot, Netherlands) mounted in a computer controlled filter wheel (Tofra, Inc., Palo Alto, California, USA) were positioned between the LED and the aquarium to control the light intensity. To make the light path in the raceway collimated, a Fresnel lens (95 mm × 135 mm, optical PVC, 3Dlens.com, Taiwan) was attached one focal length from the LED (12 cm). The LED, filter wheel, and Fresnel lens were assembled in a
have been shown to be correlated to the fjord depth distribution and the direction of phototaxis in C. f inmarchicus.15 Individuals with low lipid reserves were more likely to be present in the surface layer in daytime and to display positive phototaxis than individuals with larger lipid reserves; thus it is proposed that the need for food overrides the tendency to migrate downward to decrease the risk of predation.15 As a key species, C. f inmarchicus has been extensively studied, in particular ecological aspects such as geographical distribution, and seasonal and daily depth migration patterns.5,13,16 The species has also been used in a range of ecotoxicological studies investigating the effects of contaminant exposure on gene expression and metabolites, in addition to acute toxicity.17−20 In several of these studies lipid reserves have been shown to influence the effect of the contaminant exposure; for example, small lipid storage appeared to reduce survival during oil exposure.17,18 The aim of the present study was to investigate the effects of exposure to sublethal concentrations of the water accommodated fraction (WAF) of fresh crude oil on the phototactic behavior of copepodite stage 5 (C5) of C. f inmarchicus, which is the final stage prior to reaching maturity. As responses to light are controlling the diel vertical migration dynamics and hence the subsequent copepod distribution in the water column, effects on the phototactic response, for example from oil emissions, could alter the migration pattern. The migration and depth distribution pattern associated with diel vertical migration is considered a prerequisite for sufficient feeding and survival and hence the ecological success of the species, and a change in the diel vertical migration could accordingly have consequences on the population and community levels.
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MATERIALS AND METHODS Copepod Culture. Experimental copepods were collected from the continuous C. f inmarchicus culture at SINTEF/ NTNU Centre of Fisheries and Aquaculture (Trondheim, Norway). The culture was established from copepods collected in Trondheimsfjorden, Norway in October 2004.21 At the time of the experiments (August 2011), the copepod culture had been running for 30 generations under laboratory conditions. The culture is reared on a mixture of unicellular algae Rhodomonas baltica Karsten, Isochrysis galbana Parke, and Dunaliella tertiolecta Bucher, and maintained in running seawater in polyester containers (280 L) at ∼10 °C. The culture is kept in a set light-dark cycle regime of 18:6 h, with 6 h of dawn and 6 h of dusk in the first and last part of the light period, respectively. This corresponds to light conditions in late April at 63°N. Generation of Exposure Solutions. Fresh low sulfuric naphtenic North Sea crude oil of medium density from the Troll B production field in the North Sea (60°39′ N, 3°44′ E) was chosen for the study. The water accommodated fraction (WAF) was generated according to Croserf methodology with slow stirring in order to limit oil droplets in the solution.22 A baked glass bottle (10 L) was filled with 9 L of filtered (0.22 μm, Sterivex, Millipore, MA, USA) natural seawater collected at 70 m depths in the bottom current of oceanic water in Trondheimsfjorden. Oil (1.0 ± 0.1 g) was carefully added onto the surface with a syringe alongside a glass tube extending down into the water, giving a loading close to the desired 1:10 000 oil/water ratio. Low energy vortex-free magnetic stirring was applied for 96 h at 10 °C before the water phase was siphoned off, without disturbing the oil film on the surface, and 14427
dx.doi.org/10.1021/es4037447 | Environ. Sci. Technol. 2013, 47, 14426−14433
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Wetzlar, Germany), and controlled by the software Fire-i v.3.01.0.111 (Unibrain Inc., San Diego, USA) operated from a computer. The copepods were anesthetised with tricaine methanesulfonate (Finquel, Argent Laboratories, Redmond, USA; 1.5 g/L stock solution in seawater) and oriented in a fixed position under the microscope prior to photographing. Biometric analyses of the copepods were conducted manually (ImageJ 1.43u software25) with the aid of a graphic tablet (Cintiq 12wx, Wacom Co. Ltd., Japan). The software was set to scale by measuring an image of a calibration slide (E. Leitz, Wetzlar, Germany) captured at the same magnification as the copepods. The volumes of lipid sac and body length were calculated on the basis of the length of prosome and the lipid sac, and the corresponding areas of these two compartments.26 Lipid sac percent is lipid sac volume relative to total prosome volume. To reduce handling stress on the copepods prior to experiments, experimental C5 copepodites were sorted out of the culture by eye, but this method may result in a low percentage of misidentified adults in the selected cohort. In additions, some individuals presumably moulted during the exposure time. Thus, the developmental stage (C5 and adults) and sex (adult males and females) were re-examined after the exposure based on morphological criteria.2 Chemical Analysis. Exposure media for chemical analysis (approximately 800 mL) were collected from the undiluted WAF before the start of the exposure and from the exposure solution at the end of the exposure period (96 h). The analytical program included total extractable organic material (TEOM), volatile components (VOC), and semivolatile components (SVOC). Acidified water samples were extracted with dichloromethane (DCM), dried over Na2SO2, and concentrated to 1 mL. Determination of the total extractable organic compounds (C10−C36) was performed on the DCM extracts by gas chromatography−flame ionization detector (GC-FID). The system comprised an Agilent 6890N GC fitted with an Agilent 7683B series autosampler. The column was an Agilent J&W HP-5 fused silica capillary column (30 m × 0.25 mm ID × 0.25 μm film thickness). The carrier gas was helium at a constant flow of 1.5 mL/min. Samples of 1 μL were injected into a 310 °C split/splitless injector. The oven was heated to 40 °C for 1 min, and then the heating was ramped up to 315 °C at a rate of 6 °C/min, before the temperature was held at 315 °C for 15 min. Analysis for SVOCs including phenols, naphthalenes, and three- to five-ring polycyclic aromatic hydrocarbons (PAHs) were performed on DCM extracts by GC−mass spectrometry (GC−MS) operated in selected ion monitoring mode. The system comprised an Agilent 6890N GC with an Agilent 5975B quadrupole mass selective detector (MSD). The column was an Agilent J&W HP-5MS fused silica capillary column (60 m × 0.25 mm i.d. × 0.25 μm film thickness). The carrier gas was helium at a constant flow of 1.2 mL/min. A 1 μL sample was injected into a 310 °C split/splitless injector. The oven was heated to 40 °C for 1 min, then heated to 315 °C at 6 °C/min and held for 15 min. Data and chromatograms were monitored and recorded using MSD ChemStation (version D.03.00.611) software. The MSD ion source temperature was 230 °C. A total of 34 targeted VOCs (C5−C10) were determined by purge and trap gas chromatography/mass spectrometry, using a modified US Environmental Protection Agency EPA-Method 8260, with a 50 m (0.20 mm ID, 0.50 μm film thickness) Supelco Petrocol capillary column. Target analyses were
single tailored light-proof unit to avoid stray light from the LED. The phototactic behavior of the copepods was recorded using a video camera (Sony Handycam HDR-XR520-VE, Sony) placed perpendicular to the aquarium on a quadrapod stand (Quadrapod Elite Copy Stand, Forensic Imaging, Inc., US). The aquarium was positioned on a table with a 48 cm × 48 cm opening for near-infrared illumination (near-infrared lamps, Eneo, Germany with Kodak Wratten No. 87C filters, Edmund Optics Ltd., York, UK with 0% transmission up to ∼790 nm wavelength) from below to provide light for video recording. The setup was enclosed in a custom-made black fabric cape. The experimental setup is described in more detail in Miljeteig et al.21 Phototaxis Experiments and Analysis. The exposure bottles were kept in darkness for dark acclimation for a minimum of one hour prior to the behavioral experiments. All experiments were conducted with copepods in the same diurnal phase, i.e. in daytime between 0800 and 1700 h. The 60 copepods were transferred from the exposure bottle to clean seawater and then to the aquarium raceway, using only red light to minimize light exposure. The copepods were randomly distributed in the raceway and kept in darkness for 10 min before the light stimulus was switched on. The light stimulus covered three subsequent periods (each lasting 10 min) of low but increasing irradiance (0.99 × 10−6, 9.8 × 10−6, and 83 × 10−6 μmol photons m−2 s−1), including the irradiance level that has previously been shown to evoke a phototactic response (9.8 × 10−6 μmol photons m−2 s−1) in the C5 of the species.24 The light exposure regime was hence arranged to reveal even minor deviations in light sensitivity between the groups. The movements of the copepods during the dark period and the three irradiance levels were recorded on HD video. The positioning and change in distribution of the copepods relative to the light stimulus in the raceway in the test aquarium were analyzed using one image frame per minute extracted from the video, providing a total of 10 images representing each level of irradiance. The images were processed and analyzed with ImageJ software25 as described in more detail in Miljeteig et al.24 In short, the images were processed to improve contrast and remove stationary objects (edges, air bubbles, etc) before an automated particle analysis was conducted to determine the position of the 60 copepods relative to the light stimulus in each image frame. To group the copepods for biometric analysis, the raceway was divided into three sections by inserting two walls after 10 min at the third and highest irradiance level. Copepods in the volume closest to the light emission source, corresponding to 20% of the total volume in the experimental raceway, were defined to show positive phototaxis. Accordingly, copepods in the volume furthest from the light stimulus source, also corresponding to 20% of the total volume in the raceway, were defined to show negative phototactic response. The remaining copepods in the midsection, corresponding to 60% of the volume in the experimental raceway, were defined to show no phototactic response. The copepods in each section were transferred to 1L beakers and kept at a temperature of 10 (±2) °C until further processing for images for biometric analysis within 36 h. Images of the copepods for biometric analyses were captured at fixed magnifications with a still-video camera (Sony DFWSX900, Sony Corp, Japan), mounted on a dissecting binocular microscope (Leica MZ125, Leica Microsystems GmbH, 14428
dx.doi.org/10.1021/es4037447 | Environ. Sci. Technol. 2013, 47, 14426−14433
Environmental Science & Technology
Article
Table 1. Exposure Concentrations (μg/L) of the Main Component Groups in Water Accommodated Fraction from Fresh Crude Oil at the Start of the Exposure Experiments and at the End of the Longest Exposure Series (96 h) for Each Exposure Rounda exposure 1
exposure 2
start
end (96 h)
start
end (96 h)
mean
SD
naphthalenes two- to three-ring PAH four- to six-ring PAH T-PAH
18.76 1.36 0.022 20.14
19.36 1.13 0.014 20.50
18.47 1.33 0.022 19.83
19.37 1.14 0.010 20.52
18.99 1.24 0.017 20.25
0.45 0.12 0.006 0.33
VOC C5−C9 GC/FID C10−C36 TPH
255.5 74.9 330.3
198.2 86.5b 284.7
268.2 73.7 341.9
206.5 75.5 282.0
232.1 77.6 309.7
34.9 6.0 30.9
a
All values are mean of duplicate samples. Start concentrations are based on initial equilibrium WAF divided by the dilution factor used. End concentrations are direct measurements. Total petroleum hydrocarbon (TPH) was calculated by summing the concentrations of volatile organic compounds (VOC; C5−C9) and the total extractable organic compounds (GC/FID; C10−C36). Total polycyclic aromatic hydrocarbon (T-PAH) concentrations were calculated as the sum of naphthalenes (C0−C4) and two- to six-ring PAHs. SD is standard deviation. bOne value (124.0) excluded as a significant outlier according to Grubbs test (P < 0.01).
statistical analyses, model selection through AIC (Supporting Information, Table S1) gave a best fit model containing the following predictor variables: light level, exposure, and the interaction term between the two variables (Table 2). Exposure
detected with an Agilent 5973B MSD and the data were acquired using the Agilent EnviroQuant Chemstation software. Total petroleum hydrocarbon (TPH) was calculated by summarizing the concentrations of volatile organic compounds (VOC; C5−C9) and the total extractable organic compounds (GC/FID; C10−C36). Total PAH (T-PAH) concentrations were calculated as the sum of naphthalenes (C0−C4) and twoto six-ring PAHs. Statistical Analysis. Differences in phototactic behavior between exposed and unexposed (control) copepod groups were analyzed using binomial generalized mixed effect models with replicate as a random factor with random intercept. The best model was selected using Akaike’s Information Criterion (AIC) from combinations of the variables light level, exposure, and exposure time and interaction terms between the variables. The copepod distribution at the end of each irradiance step was used for further statistical analysis. Control and exposed groups were tested pairwise. The significance level was set to α = 0.05. All statistical analyses were conducted in the R environment27 with the packages nlme28 and lme4.29
Table 2. Best Fit Model Based on AIC with Binomial Generalized Mixed Effect Models with Replicate as Random Factor and with Random Intercept for Phototactic Behaviour in C. finmarchicus Exposed to Water Accommodated Fraction of Fresh Crude Oil for 24, 48, 72, and 96 ha
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RESULTS AND DISCUSSION The phototactic behavior was investigated in C. f inmarchicus C5 exposed to a sublethal concentration of WAF of fresh crude oil for 24, 48, 72, and 96 h, and no mortalities were observed. The average of exposure concentrations based on analysis before and after exposure was 310 ± 32 μg/L total hydrocarbon (TPH; mean ± standard deviation [SD]) corresponding to 20.25 ± 0.33 μg/L total PAH (mean ± SD; Table 1). During the 96 h exposure the TPH concentration dropped by approximately 16%. This drop was almost exclusively caused by loss of volatile components, presumably by biological degradation since the exposure bottles were closed during the entire exposure period. Although the T-PAH appears to be constant, there was a reduction of almost 50% of the four- to six-ring PAHs. This is assumed to be caused by a combination of attachment to surfaces and biological uptake. The loss of the various PAH groups correlates with their lipophilic properties related to increased bioaccumulation, which increases from naphthalene toward the four- to six-ring PAHs. Napthalenes constitute about 93% of the T-PAH and are not depleted, thus the TPH concentration was fairly constant. The exposure to WAF of fresh crude oil significantly influenced the phototactic behavior of C5 C. f inmarchicus. For
fixed effects
estimate
std. error
z value
P value
intercept exposure light level 8.3 × 10−6 light level 9.8 × 10−6 light level 9.9 × 10−6 exposure: light level 8.3 × 10−6 exposure: light level 9.8 × 10−6 exposure: light level 9.9 × 10−6
−0.0270 0.0345 −0.3434 −0.3411 −0.1501 −0.6993 −0.2133 0.1022
0.0677 0.0749 0.0755 0.0755 0.0750 0.1100 0.1073 0.1060
−0.40 0.46 −4.55 −4.52 −2.00 −6.36 −1.99 0.96
0.691 0.645
