Ecotoxicology DOI 10.1007/s10646-014-1268-9

Active avoidance from a crude oil soluble fraction by an Andean paramo copepod Cristiano V. M. Arau´jo • Matilde Moreira-Santos Jose´ P. Sousa • Valeria Ochoa-Herrera • Andrea C. Encalada • Rui Ribeiro



Accepted: 22 May 2014 Ó Springer Science+Business Media New York 2014

Abstract Several oil spills due to ruptures in the pipeline oil systems have occurred at the Andean paramo. A sample of this crude oil was mixed with water from a nearby Andean lagoon and the toxicity of the soluble fraction was assessed through lethal and avoidance assays with a locally occurring copepod (Boeckella occidentalis intermedia). The integration of mortality and avoidance aimed at predicting the immediate decline of copepod populations facing an oil leakage. The 24-h median lethal PAH concentration was 42.7 (26.4–91.6) lg L-1. In the 12-h avoidance assay, 30 % avoidance was recorded at the highest PAH concentration (19.4 lg L-1). The mortality at this PAH concentration would be of 25 % and, thus, the population immediate decline would be of 55 %. The inclusion of non-forced exposure testing with the quantification of the avoidance response in environmental risk assessments is, therefore, supported due to underestimation of the lethal assays. Keywords Ecuador  Paramo  Non-forced exposure  Oil spill  Polycyclic aromatic hydrocarbons

C. V. M. Arau´jo (&)  M. Moreira-Santos  J. P. Sousa  A. C. Encalada  R. Ribeiro IMAR-Instituto do Mar, Department of Life Sciences, University of Coimbra, Apartado 3046, 3001-401 Coimbra, Portugal e-mail: [email protected] C. V. M. Arau´jo Departamento Central de Investigacio´n (DCI), Universidad Laica Eloy Alfaro de Manabı´ (ULEAM), Ciudadela Universitaria, vı´a San Mateo, Manta, Ecuador V. Ochoa-Herrera  A. C. Encalada Universidad San Francisco de Quito, Cole´gio de Ciencias Biolo´gicas y Ambientales, Diego de Robles y Vı´a Interocea´nica, 17-1200-841 Quito, Ecuador

Introduction Classical experiments in aquatic ecotoxicology normally consist in exposing organisms to different concentrations of a contaminant or different dilutions of a sample under forced conditions, determining thus the degree of contamination to which the organisms are permanently exposed and assuming that organisms in the field respond passively to contamination (Roberts et al. 2008; Arau´jo et al. 2012). Although forced exposure to a given contaminant concentration is the scenario most commonly used in environmental risk assessment, it ignores the displacement of the organisms to less contaminated zones, as expected in real conditions. Therefore, forced exposure often represents an unrealistic scenario of contamination, lacking ecological relevance for species that are able to move and avoid contaminants (Lopes et al. 2004; Moreira-Santos et al. 2008; Rosa et al. 2008, 2012; Arau´jo et al. 2012, 2014). A nonforced exposure-an avoidance assay-checks the organisms’ escape from contamination. The use of avoidance as an assay endpoint for actively moving organisms has been considered a relevant and valuable additional tool in ecotoxicology (Lopes et al. 2004; Moreira-Santos et al. 2008; Rosa et al. 2008, 2012; Dornfeld et al. 2009; Arau´jo et al. 2012, 2014). Moreover, the integration of avoidance with mortality data allows the prediction of the population immediate decline (PID) at the impacted ecosystem (Rosa et al. 2012). So far, results of avoidance assays obtained with amphipods (Kravitz et al. 1999), cladocerans (Lopes et al. 2004; Rosa et al. 2008, 2012), fish (Svecevicˇius 1999; Svecevicˇius 2001; Wells et al. 2004; Moreira-Santos et al. 2008), gastropods (Arau´jo et al. 2012), and tadpoles (Arau´jo et al. 2014) have proved to be a promising complementary tool, solving problems related to the forced exposure of aquatic organisms in toxicity testing.

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Avoidance from contamination can occur at concentrations lower and/or earlier than lethal and other sub-lethal effects (feeding, growth, reproduction) (Lopes et al. 2004; Rosa et al. 2008; Owojori and Reinecke 2009). At the ecosystem level, the avoidance of all the individuals of a given species from contamination is an unequivocally ecologically relevant effect because it corresponds to the extinction of that population (Lopes et al. 2004). If avoidance occurs at partially lethal concentrations, then ignoring it will underestimate the PID (Rosa et al. 2012; Arau´jo et al. 2014). When sub-lethal responses (e.g. growth, reproduction) are measured under a forced exposure, extrapolations from the effects on individuals up to the ecosystem level are time-delayed as deleterious effects and population downsizing tend to be gradual over time (Rosa et al. 2012). In contrast, avoidance has direct and instantaneous effects on ecosystem structure and function, as the organisms’ disappearance is immediate (Roberts et al. 2008; Rosa et al. 2012). Therefore, if the avoidance response occurs at concentrations similar to those producing other sub-lethal effects, immediate consequences of avoidance make it a more pertinent measurement than other sub-lethal endpoints, since it leads, as mortality, to a decrease in the population size in the short-term (Lopes et al. 2004; Moreira-Santos et al. 2008; Rosa et al. 2008, 2012; Arau´jo et al. 2014). Paramos are high altitude tropical ecosystems (between 3,000 and 4,500 m a.s.l.) that are composed mostly of grassland and scrubs, being recognizably vulnerable environments due to extreme environmental conditions such as low temperature, low atmospheric pressure, high humidity and high solar radiation (Sarmiento 1986; Ortiz et al. 2005). In the particular case of the Northern Andes (Venezuela, Colombia, Ecuador, and Northern Peru), special attention has to be deserved to the relevant role that high altitude ecosystems have in the storage of water and in the regulation of hydrological flows (Farley et al. 2004; Buytaert et al. 2006). In fact, despite the isolated and fragmented occurrence of the paramo over the Andean highlands, it has been estimated that this ecosystem provides water for irrigation, hydropower generation and human consumption for at least 12 million people across Northern Andes, constituting more than 80 % of the total water supply for large capital cities such as Bogota´ and Quito (Josse et al. 1999; Aveiga del Pino et al. 2005). During the past two decades, paramo ecosystems across the world have received special attention partially due to the ever-increasing threats they are experiencing (e.g. oil spills and deforestation), but also to their crucial role in the regulation of important ecosystem services such as carbon storage, soil stabilization and water supply (Viviroli et al. 2003; Messerli et al. 2004; Viviroli and Weingartner 2004). Excessive nutrient inputs, pesticide use, burning, pasture,

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oil and gas transportation have been described as the most serious environmental problems in paramo regions (Ortiz et al., 2005; Finer et al., 2008). Unfortunately, contamination by crude oil-a wide world problem for several terrestrial, marine and freshwater ecosystems-has been also recorded in the Ecuadorian paramo (Aveiga del Pino et al. 2005; Finer et al. 2008; Kelsh et al. 2009; Sua´rez et al. 2009). Since the 1970s, oil is considered a key element in the Ecuadorian economy, with the extraction of around two billion barrels of crude oil a year already from the Ecuadorian Amazon (Finer et al. 2005), which is associated to accidental discharges of toxic wastes into the land and waterways (Sebastia´n and Hurtig 2005). As consequence, a great controversy has been generated regarding the process of oil exploration and extraction as well as its impacts on the environment and its potentially adverse effects on health (Kelsh et al. 2009). In 2003, a breakage in the transEcuadorian oil pipeline system generated a flow of 22,000 petroleum barrels which contaminated soil, two streams (Tambo and Sucus) and the Papallacta Lake (Napo province, NE of Quito), where total petroleum hydrocarbon reached 6,400 mg L-1 (Aveiga del Pino et al. 2005). Although environmental impacts have not been satisfactorily documented, the biological communities were almost extinct and this scenario led to a hard decontamination process to reduce the impact in lake and river waters, sediment, rocks, and surrounding vegetation (Aveiga del Pino et al. 2005). The present study aimed at assessing the ability of an Ecuadorian paramo autochthonous copepod, Boeckella occidentalis intermedia, to actively avoid a contamination gradient of the soluble fraction of the crude oil being transported in the trans-Andean pipeline. A test system with non-forced exposure was used to measure the avoidance response and it was integrated with mortality to predict the PID (Rosa et al. 2012).

Materials and methods Test organisms The copepod Boeckella occidentalis intermedia, which is abundant in oligothrophic Ecuadorian lakes (Torres and Rylander 2006), was used as test species. Organisms were collected from an Ecuadorian paramo lagoon (Laguna del Amor), located inside the Cayambe Coca National Park and very near the oil pipelines (0°9´31.14S; 78°11´45.18W, altitude of 4,209 m a.s.l., pH of 6.54, conductivity of 9.41 lS cm-1, and temperature around 10 °C), using a gently trawled 50-lm mesh plankton net. After collection into polyethylene terephthalate bottles, 2–3-mm long adults were immediately transported to the laboratory, in

Active avoidance from a crude oil soluble fraction Table 1 Concentrations and proportions of polycyclic aromatic hydrocarbons (PAH; analysed by gas chromatography linked to mass spectrometry) in the sample of crude oil soluble fraction prepared from a raw sample of crude oil transported through Ecuadorian transAndean pipelines Compounds

Concentration (lg L-1)

Proportion (%)

Acenaphthene

\0.01

\0.005

Acenaphthalene

4.10

Anthracene

2.00

1.05

Benzo(a)anthracene

3.60

1.86

Benzo(gui)perylene

0.52

0.27

Benzo(a)pyrene

0.52

0.27

Benzo(b)fluoranthene

0.50

0.26

Benzo(k)fluoranthene

0.31

0.16

Chrysene

3.00

1.57

Dibenzo(a,h)anthracene Fluoranthene

0.35 1.00

0.18 0.52

Fluorene Indeno(1,2,3-cd)pyrene Naphthalene Phenanthrene Pyrene Total-PAH

10.7 0.29 111 53.0 2.60 194

2.13

5.52 0.15 57.22 27.32 1.32 100

thermally insulated boxes (at around 10 °C) and maintained in cultures until use in assays (within 4 days). Only 2–3-mm long adults were used in assays to facilitate handling and counting, and to eliminate possible differences in sensitivity due to life stage and females carrying eggs were discarded. Cultures were maintained at 23–24 °C and 12/12 h (light/dark) photoperiod, in water from Laguna del Amor (renewed every other day). No food was provided during culturing. Mortality in laboratory cultures was below 10 %. The crude oil soluble fraction A raw sample of crude oil, gently provided by the REPSOL company, belonging to the same batch transported through Ecuadorian trans-Andean pipelines was used as contaminant. A 10 % (v/v) suspension with the same site water from where the organisms were collected was prepared. This suspension was vigorously shaken for 15 min and left to rest for 2 h in darkness to allow phase separation. Then, the supernatant was siphoned and used for assays as the crude oil water-soluble fraction, which intended to represent the bioavailable water-soluble fraction of this suspension. It was stored at 4 °C in darkness for polycyclic aromatic hydrocarbons (PAH) analysis (Table 1), by gas chromatography linked to mass spectrometry in a certified laboratory (LQA-Ambiente, Lisbon, Portugal).

The avoidance assay system The system for avoidance assays was similar to that developed by Rosa et al. (2012). Each test chamber was 2.8-m long and consisted of seven interconnected compartments (Fig. 1). Each compartment (length: 40 cm; central diameter: 7 cm; diameter in the extremities: 2.1 cm; upper opening: 14 9 4 cm) was constructed with two 500-mL polyethylene terephthalate bottles. A white glue (Sikaflex-11FC?, Baar, Switzerland) was used to construct each compartment and subsequently to connect them. Each compartment had a total volume of approximately 700 mL, but was filled with 600 mL of test solution, making a total chamber volume of 4200 mL (600 mL 9 7 compartments). Initially, the avoidance system was tested to verify the stability of the contamination gradient. A sodium chloride solution in tap water was chosen for this purpose. A 100 mg L-1 solution (considered 100 %) was serially diluted with tap water to obtain five concentrations (16, 33, 50, 66 and 83 %) plus a control (0 %). Before introducing the NaCl solutions, compartments were isolated with plasticine spheres wrapped with parafilm to avoid mixing between adjacent compartments. Afterwards, a volume of 600 mL of each of the seven solutions was cautiously poured into each compartment. The plasticine spheres were removed and the system was left to equilibrate for approximately 2 h after which the evaluation of the stability of the linear concentration gradient was initiated. The procedure to verify the system stability was based on conductivity measurements, which were recorded at 2 h (after the stabilization period), considered the initial reading, and at 12 h (maximum exposure time). Conductivity was used as it was shown to be a reliable indicator of NaCl concentrations (r2 = 0.9991, n = 13). The possible mixture of solutions along the test system did not alter the initial NaCl concentrations in each compartment (100, 83, 67, 50, 33, 17, and 0 % of 100 mg L-1), since no significant differences were found between expected (i.e., initial) and observed concentrations at 12 h (X26 = 2.47, p = 0.87). Lethal and avoidance assays A lethal assay was performed with six dilutions of the crude oil water-soluble fraction: 2.5, 5, 10, 20, 30, and 40 % (corresponding to 5, 10, 19, 39, 58, and 78 lg L-1 of total PAH, respectively), plus the culture medium as control (0 %). Each concentration was tested in duplicate with four organisms per replicate. The assay lasted 24 h and was carried out under the same temperature and light regime of the cultures.

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Fig. 1 Scheme of a single compartment and of the seven-compartments assay chamber with non-forced exposure to a stable and linear contamination gradient (100, 83, 67, 50, 33, 17, and 0 % of the

contaminant) to evaluate the active spatial avoidance by the copepod of a high altitude Ecuadorian paramo lagoon, Boeckella occidentalis intermedia

After verifying the gradient stability in the avoidance system, test chambers were vigorously washed and then filled with dilutions of the crude oil water-soluble fraction, using the culture medium (water from Laguna del Amor): 1, 0.83, 0.67, 0.5, 0.33, 0.17, and 0 % (corresponding to 19.4, 16, 13, 9.7, 6.4, 3.3, and 0 lg L-1 of total PAH, respectively). To attest that the copepods distribution was random along the compartments of the test system (with no influence of non-controlled experimental factors and no preference for any compartment), three replicated control chambers with culture medium only in all compartments were used. In the avoidance assay with the contamination gradient four replicates were used. Ten copepods were introduced in each compartment (70 in total for each test chamber). Organisms’ distribution along compartments was checked after a 12 h exposure, immediately after closing the connections between compartments with the plasticine spheres, to minimize organisms’ displacement during counting. Assays were performed under darkness and controlled temperature of 23–24 °C.

number of copepods placed in that compartment at the start of the assay (Moreira-Santos et al. 2008). For the remaining concentrations, the number of expected copepods includes the organisms placed initially in the respective compartment plus the organisms placed in the adjacent compartments with higher concentrations (Moreira-Santos et al. 2008). As each compartment had 10 copepods, expected organisms in the highest concentration would be 10, while for the second, third, a.s.o. concentrations, expected copepods would be 20, 30, a.s.o., respectively. As survival in control and PAH-contaminated chambers was similar (93 and 90 %, respectively), a uniform mortality among compartments was assumed and, therefore, the number of expected organisms in each compartment of the gradient chambers was reduced by 10 %. The 12-h AC20 (concentration that triggers avoidance of 20 % of exposed organisms) and the respective 95 % CI were determined using the software PriProbit 1.63 (Sakuma 1998). The PID (in %) induced by PAH was calculated for each concentration used in the avoidance assay through the integration of results from the 12-h avoidance and the 24-h lethal assays. The PID is the proportion of the avoiders plus the proportion of the non-avoiders that would die, which equals the proportion of the dead ones plus the proportion of the survivors that would avoid. The PID20 and PID50 values and associated 95 % CI were also calculated using the software PriProbit 1.63 (Sakuma 1998). It should be emphasized that, because as recommended in international guidelines (e.g. OECD 1998), regression-based estimations (i.e., LCx, ACx and PIDx) were the objective of the present study, a higher number of concentrations replicated once in time was favoured against the use of replication in time.

Data analysis In the 24-h lethal assay, the median lethal concentration (i.e., the interpolated concentration killing 50 % of the organisms) and the respective 95 % confidence interval (95 % CI) were determined using the software PriProbit 1.63 (Sakuma 1998), as well as the LC20. The randomness of organisms’ distribution among compartments within chambers of the avoidance system when exposed exclusively to control water for 12 h was checked with the Kruskal–Wallis’ test. In the avoidance assay with crude oil, the number of avoiders was computed as the number of expected organisms minus the number of observed organisms, and the avoidance percentage in each compartment as the number of avoiders divided by the expected ones. If survival is 100 %, then expected organisms in the compartment with the highest PAH concentration would be the

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Results The 24-h median lethal total PAH concentration for B. occidentalis intermedia was of 43 lg L-1 (95 % CI

Active avoidance from a crude oil soluble fraction 60 Avoidance (%) Mortality (%) Population immediate decline

Proportion (%)

50 40 30 20 10 0 3.1

6.4

9.7

13

16

19

-1

Total PAH concentrations (µg L ) Fig. 2 The observed avoidance after 12 h of exposure, the 24-h interpolated mortality (in both n = 4) and their integration-the population immediate decline (PID)-for the paramo copepod Boeckella occidentalis intermedia exposed to total PAH present in the water-soluble fraction of crude oil transported by pipelines through the Ecuadorian Andes

26–92). The interpolated mortality values at the concentrations used in the avoidance assay are presented in Fig. 2. In the absence of the contaminant, the randomness of the spatial distribution of copepods among the compartments, after 12 h, was attested (p [ 0.05). The latter, along with the maintenance of stable contamination gradient, validates the avoidance assay. Avoidance after 12 h of exposure and 24-h interpolated mortality were similarly sensitive, with almost superimposed concentration–response curves, though both only exceeding 20 % at total PAH concentrations close to 19 lg L-1 (Fig. 2): the LC20 and AC20 values were 16 lg L-1 (95 % CI 14–20) and 17 lg L-1 (95 % CI 14–21), respectively. However, together they would promote a population immediate decline above this threshold at concentrations as low as 10 lg L-1 and of 48 % at 19.4 lg L-1 (Fig. 2). Values of PID20 and PID50 were 9.9 lg L-1 (95 % CI 8.7–11) and 20 lg L-1 (95 % CI 18–25).

Discussion The observed contamination pattern of linearity and stability, using sodium chloride, validated the system for the avoidance assay. A similar gradient pattern was previously reported by Lopes et al. (2004) and Moreira-Santos et al. (2008), through the use of peristaltic pumps, and by Rosa et al. (2012) and Arau´jo et al. (2014) with no pumps, as in the present study. Even if, in a hypothetical scenario of a PAH aquatic contamination following a crude oil spill from

the trans-Andean pipeline, a vertical and/or horizontal water column stratification would occur, the ecological relevance of using a bi-compartmented system, instead of a multi-compartmented exposure one, could be questioned. This is so because it is hardly conceivable that the gradient of the crude oil water-soluble fraction would be so sharply abrupt. It is more plausible a much larger interface between the full and the zero soluble fraction. Here, the 2.8-m wide margin of a 28-m long interface ended up to be simulated, by using a system with a length of 2.8 m and a constant linear contamination gradient between 10 and 0 % of the soluble fraction. The spatial PAH gradient of 7 lg L-1 m-1 here tested (19.4 lg L-1 over 2.8 m) was well above the copepod 12-h lowest observed effective gradient (LOEG)-minimum spatial relative difference of contamination being detected and avoided-because an avoidance of 30 % was found. As discussed by Rosa et al. (2012), the LOEG is expected to be time-specific, decreasing with exposure duration. Therefore, a 12-h avoidance assay can not encompass the spatial and temporal dynamics of an oil spill and most probably underestimates the severity of effects in real field situations, but it remains as a pragmatic, though relevant, as possible approach. So, the present study is another step towards the standardization of non-forced exposure tests, contributing to fill the knowledge gap on avoidance from contaminants which pertinence has already been highlighted (Moreira-Santos et al. 2008). Avoidance was a noticeable response used by copepods when faced with a PAH contamination gradient. Thirty percent of the copepods avoided the compartment with a 10 % dilution of the crude oil soluble fraction and a clear concentration-dependent relationship was recorded. From an ecological point of view, this effect can have consequences as severe as mortality for ecosystem structure and function. Although the decrease in organisms’ abundance in the short-term has often been exclusively related to lethal effects, avoidance has the same ecological implications than mortality (Fleeger et al. 2003; Lopes et al. 2004). So, both avoidance and mortality play an important role for the PID. Integrating both is, thus, needed to best predict short-term effects. If those responses were independently treated, the risk of the population decline would be seriously underestimated. In the static test, the 24-h mortality at the 19.4 lg L-1 PAH concentration was 25 %, but, at the same concentration, avoidance reached 30 %. Thus, the population immediate decline was 55 %. As far as the authors are aware of, this was the first time copepods were shown to actively swim from contamination, as already known for another zooplankton group-the cladocerans (Lopes et al. 2004; Rosa et al. 2008, 2012). Furthermore, the present study further emphasized the probable underestimation of the risk of immediate population decline and extinction if only lethal tests, under a

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forced exposure, are used. Avoidance by a copepod of the Andean paramo was shown to be a clear and rapid response when exposed to a gradient of PAH, doubling the estimate of disappearance risk of local populations if only mortality was evaluated. Acknowledgments This study was partially funded by the Master Program in Ecology and the Laboratory of Aquatic Ecology at the Universidad San Francisco de Quito (Ecuador) through the European Master Applied Ecology (EMAE) consortium and by the Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT, Portugal) though a postdoctoral fellowship (reference SFRH/BPD/74044/2010) to CVM Arau´jo and Cieˆncia 2007-Human Potential Operational Program (POPH) and Quadro de Refereˆncia Estrate´gico Nacional (QREN) through European Social Fund (FSE) and Ministry of Education and Science (MEC) funds. We acknowledge the help in the lab and in the field of Natalia Garcı´a and Maja Celinscak. Graphical assistance in Fig. 1 was provided by FR Diz. Conflict of interest of interest.

The authors declare that they have no conflict

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Active avoidance from a crude oil soluble fraction by an Andean paramo copepod.

Several oil spills due to ruptures in the pipeline oil systems have occurred at the Andean paramo. A sample of this crude oil was mixed with water fro...
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