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Multiple stress effects on marine planktonic organisms: Influence of temperature on the toxicity of polycyclic aromatic hydrocarbons to Tetraselmis chuii
Journal of Sea Research 72 (2012) 94–98
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Multiple stress effects on marine planktonic organisms: Influence of temperature on the toxicity of polycyclic aromatic hydrocarbons to Tetraselmis chuii L.R. Vieira ⁎, L. Guilhermino CIIMAR—Interdisciplinary Centre of Marine and Environmental Research, Laboratory of Ecotoxicology and Ecology, University of Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal ICBAS—Institute of Biomedical Sciences of Abel Salazar, University of Porto, Department of Populations Studies, Laboratory of Ecotoxicology, Largo Prof. Abel Salazar, 4099-003 Porto, Portugal
a r t i c l e
i n f o
Article history: Received 29 May 2011 Received in revised form 6 February 2012 Accepted 7 February 2012 Available online 15 February 2012 Keywords: Plankton Global Warming PAHs Oil Spills Multiple Stressors
a b s t r a c t In the present context of global warming and increasing long-range transport of oil and goods by sea potentially resulting in oil spills, more knowledge on the toxicological interactions between temperature and oil components on marine organisms is urgently needed. Therefore, the effects of temperature increase on the toxicity of three polycyclic aromatic hydrocarbons (PAH; anthracene, phenanthrene and naphthalene) to the marine planktonic algae Tetraselmis chuii were investigated under laboratory conditions. T. chuii cultures were exposed for 96 h to different concentrations of each of the test substances at both 20 and 25 °C. Effect criterion was the inhibition of culture growth assessed at 24 h intervals. All the PAHs significantly reduced T. chuii growth after 96 h of exposure with 20% inhibition concentrations between 0.052 and 1.124 mg L− 1 at 20 °C, and between 0.048 and 0.831 mg L− 1 at 25 °C. At both temperatures, the ranking, in order of decreasing toxicity based on the 50% inhibition concentration, was phenanthrene > naphthalene > anthracene. The increase of temperature by 5 °C significantly increased the toxicity of all the PAHs tested. These findings highlight the importance of considering temperature variation in the ecological risk assessment of oil and other chemical spills in the marine environment, and the need of more research on the toxic effects of multiple stressors on marine organisms. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Recent oil spills in the marine environment showed again how negative the immediate ecological and economic consequences of these events can be (Albaigés et al., 2006; Crone and Tolstoy, 2010). Long term studies on the effects of oils spill events that occurred several years ago have shown that environmental conditions play a determinant role on the degradation and bioavailability of oil components and on their toxicity to marine organisms (Fathalla and Andersson, 2011; Neff et al., 2011; Tansel et al., 2011). These findings highlight the need of improving prevention systems and ecological risk assessment procedures, and of getting more knowledge on the ecological status of vulnerable areas to be protected, on both short and long term toxic effects of oil components, and on the effects of multiple stressors on marine organisms. The interactions between temperature and oil components are of special relevance considering the present context of global warming (IPCC, 2007) and the increasing trend of long-range transport of oils and goods by sea
⁎ Corresponding author at: CIIMAR—Centro Interdisciplinar de Investigação Marinha e Ambiental, Laboratório de Ecotoxicologia e Ecologia, Rua dos Bragas, 289, 4050-123 Porto, Portugal. Tel.: + 351 223401800. E-mail address: [email protected] (L.R. Vieira). 1385-1101/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.seares.2012.02.004
which potentially raises the risk of oil spills despite the improvement of preventive regulation and measures (Martínez-Gómez et al., 2009). Among oil components, polycyclic aromatic hydrocarbons (PAHs) are particularly important from an ecotoxicological point of view due to their toxicity, since several of them or their metabolites have been found to cause genotoxic, carcinogenic, endocrine disruption and other toxic effects in marine organisms (e.g. Thomas, 1988; Wessel et al., 2010; Zheng et al., 2005, 2011). The toxicity of oils and PAHs to marine organisms has been shown to be increased by temperature (Saeed et al., 1998) and UV radiation (Bellas et al., 2008; Echeveste et al., 2011; Lyons et al., 2002; Meng et al., 2007), among other factors. In marine ecosystems, planktonic algae populations have key roles as primary producers (hereafter indicated as producers). Since they are planktonic organisms they may be particularly exposed to oils immediately after spillages of these products, to oil spots floating at the sea surface for variable periods of time, and to inputs from other sources, including continental ones. Thus, the objective of the present study was to investigate the effects of temperature increase (from 20 to 25 °C) on the toxicity of three polycyclic aromatic hydrocarbons (anthracene, naphthalene and phenanthrene) to the marine planktonic algae Tetraselmis chuii in laboratory conditions. T. chuii was selected for this study because it has been used as a representative of marine phytoplankton in previous ecotoxicological studies (e.g. Ferreira et al., 2007; Leite et al., 2011; Nunes et al., 2005; Sibila et
L.R. Vieira, L. Guilhermino / Journal of Sea Research 72 (2012) 94–98
al., 2008) and it is a native species in marine ecosystems of temperate regions. The inhibition of algae culture growth was used as effect criterion as recommended in international protocols (e.g. OECD, 2006; USEPA, 2002) to access the toxic effects of chemical substances on microalgae.
2.4. Data analysis Following the OECD guideline 201 (OECD, 2006), the T. chuii average specific growth rates (μ) per day were calculated as the logarithmic increase of biomass in each test recipient, over the entire test duration (4 days), according to the equation:
2. Materials and methods 2.1. Chemicals Tested chemicals were phenanthrene (CAS no. 85-01-8), purchased from Fluka GmbH (Buchs, Switzerland), anthracene (CAS no. 120-12-7) and naphthalene (CAS no. 91-20-3), both purchased from Sigma– Aldrich Chemical (Steinheim, Germany). All the other chemicals were acquired from Sigma–Aldrich Chemical (Steinheim, Germany). 2.2. Algae culture conditions The green unicellular marine algae T. chuii was kindly provided by the Instituto de Ciencias Marinas de Andalucía (CSIC), Spain. This species was cultured in the laboratory under standardized abiotic conditions (24 h of solar spectrum light at 20 ± 1 °C), generally following the recommendations of OECD (2006). Test medium was f/2 Guillard's medium (Guillard, 1975; Guillard and Ryther, 1962), sterilized by autoclaving (Uniclave 88 - AJC, Portugal) at 121 °C for 35 min. All cultures were routinely monitored by microscopic observation (Leica DM 2000) to assure the normal status and growth of T. chuii. 2.3. Toxicity tests Tests were carried out for 96 h in static conditions, following the OECD guideline 201 “Alga, Growth Inhibition Test” (OECD, 2006). For each tested substance, two independent tests were carried out: one at 20 °C and the other at 25 °C, in temperature controlled rooms under 24 h of light (solar spectrum lamps (Arcadia, model FO30), 90 μE/m 2/s). The distance between the source of light and test recipients was 14 cm in all treatments and tests; test recipients were never protected from light during the exposure period to simulate environmental conditions; thus, the toxicity assessed corresponds to the overall effects of parental compounds, metabolites and environmental degradation products formed during the exposure period. Individual stock solutions (500 mg L − 1) of anthracene, phenanthrene and naphthalene were prepared in acetone (100%). For each PAH, test solutions were obtained by dilution of the corresponding stock solution in f/2 medium. The nominal test concentrations were 0.09, 0.18, 0.36, 0.72, 1.44, 2.88 and 5.76 mg L − 1 for anthracene; 0.045, 0.09, 0.18, 0.36, 0.72, 1.44 and 2.88 mg L − 1 for naphthalene and 0.035, 0.07, 0.14, 0.28, 0.56, 1.12 and 2.24 mg L − 1 for phenanthrene. In each test and for each temperature, two controls were included in the experimental design: one with f/2 medium only (hereafter designated as “control”) and another one (hereafter indicated as “acetone-control”) with the highest concentration of acetone used in the corresponding PAH treatments, namely 11.52, 5.76 and 4.48 mL of acetone per L of test medium for anthracene, naphthalene and phenanthrene, respectively. Test recipients (500 mL Erlenmeyer flasks) were filled with 400 mL of filtered culture medium, with perforated rubber stoppers, equipped with aeration systems and sterilized (Uniclave 88 - AJC, Portugal) at 121 °C for 35 min prior to algae inoculation. Treatments were inoculated with a volume of exponential growing algal cultures to obtain the concentration of 1 × 104 cells mL− 1 in test medium at the beginning of the assays. In each assay, three replicates per treatment were used. Growth was determined by cell counts of samples taken from each treatment at 0, 24, 48, 72 and 96 h, using a Neubauer Improved bright-line chamber and a microscope (Leica DM 2000), after cell fixation by Lugol. During the test, medium temperature and pH were monitored every 24 h (pH meter 3310 Jenway).
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μ ij ¼
lnX j lnX i 1 day tj ti
where μi-j is the average specific growth rate from time i to j, Xi is the biomass at time i and Xj is the biomass at time j. To assess the effects of each PAH on the algae growth at each temperature and to determine the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC), different treatments were compared by one-way ANOVA, followed by Dunnett's test comparing each PAH treatment and the acetone-control against the control (Zar, 1996). Prior to ANOVA, data were tested for normality of distribution (Kolmogorov– Smirnov normality test) and homogeneity of variance (Levine's test) (Zar, 1996) and data transformations were made when necessary. For each PAH and temperature, the percent inhibition of growth rate was calculated for each treatment replicate using the equation (OECD, 2006): %I r ¼
μ c −μ t 100 μc
where %Ir is the percent inhibition in average specific growth rate; μC is the mean value for average specific growth rate (μ) in the control group since no significant differences in the average specific growth rates were found between the control and the acetone-control in any of the toxicity tests (Table S1); and μt is the average specific growth rate for the treatment replicate. The %Ir values calculated for each temperature, PAH and test replicate were transformed by probit analysis (Finney, 1971) and plotted against the logarithm of the test substance concentration to obtain the toxicity curves. From each, the 20% and 50% inhibition concentrations (IC20 and IC50, respectively) were calculated. To compare the effects of temperature on the toxicity of different PAHs, all the toxicity curves were compared using a two-way analysis of covariance (ANCOVA) (Zar, 1996) with the different exposure levels as covariates, and PAHs and temperatures as fixed factors in a full-factorial model. All statistical analyses were performed using the SPSS 17.0 software package and differences were considered statistically significant when p b 0.05. 3. Results The microalgae average specific growth rates over the period of the test (4 days), for each tested PAH, at both temperatures are provided in Supplementary Data (see Table S1). In all the assays, significant differences in average specific growth rates among differences were found at both 20 °C (anthracene: F(8,26) = 229.9, p b 0.05; naphthalene: F(8,26) = 113.5, p b 0.05; phenanthrene: F(8,26) = 145.8, Table 1 Comparison of the no observed effect concentrations (NOECs) and the lowest observed effect concentrations (LOECs) of anthracene, naphthalene and phenanthrene to T. chuii at 20 and 25 °C. 20 °C
−1
Anthracene (mg L ) Naphthalene (mg L− 1) Phenanthrene (mg L− 1)
25 °C
NOEC
LOEC
NOEC
LOEC
0.180 0.180 0.035
0.360 0.360 0.070
0.090 0.180 0.035
0.180 0.360 0.070
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Table 2 Estimated 20% and 50% growth inhibition concentrations (IC20s and IC50s, respectively) of anthracene, naphthalene and phenanthrene to T. chuii after 96 h of exposure at 20 and 25 °C. 95% confidence intervals are indicated within brackets. 20 °C
−1
Anthracene (mg L ) Naphthalene (mg L− 1) Phenanthrene (mg L− 1)
25 °C
IC20 (95%CI)
IC50 (95%CI)
IC20 (95%CI)
IC50 (95%CI)
1.124 (0.570–1.711) 0.767 (0.562–0.964) 0.052 (0.038–0.067)
3.326 (2.718–4.233) 1.813 (1.571–2.136) 1.316 (1.032–1.729)
0.831 (0.388–1.207) 0.465 (0.255–0.656) 0.048 (0.033–0.065)
2.145 (1.734–2.743) 0.992 (0.788–1.301) 0.262 (0.236–0.291)
p b 0.05) and 25 °C (anthracene: F(8,26) = 326.1, p b 0.05; naphthalene: F(8,26) = 270.5, p b 0.05; phenanthrene: F(8,26) = 447.8, p b 0.05). As indicated by the Dunnett's tests carried out for each individual PAH and temperature, no significant differences between the control and the acetone-control were found, the NOECs determined for the average specific growth rates varied from 0.035 mg L − 1 and 0.180 mg L − 1, while LOECs ranged from 0.070 mg L − 1 to 0.360 mg L − 1 for both temperatures (Table 1). The IC20 and IC50 values calculated from the toxicity curves (log concentration vs. probit transformation of %Ir values) are shown in Table 2. At 20 °C, IC20s ranged from 0.052 for phenanthrene to 1.124 mg L − 1 for anthracene, while the corresponding values at 25 °C ranged from 0.048 to 0.831 mg L − 1 for phenanthrene and anthracene, respectively. At 20 °C, the IC50s ranged from 1.316 mg L − 1 for phenanthrene to 3.326 mg L − 1 for anthracene, while at 25 °C they ranged from 0.262 for phenanthrene to 2.145 mg L − 1 for anthracene. The toxicity curves (log of PAH concentration vs. % of growth inhibition) obtained for different PAHs at both 20 and 25 °C are
shown in Fig. 1. Significant differences among the toxicity curves were found (ANCOVA: F11,111 =165.012, p b 0.05, observed power= 1.0), with statistical significant differences among PAHs (F2,111 = 109.117, p b 0.05), between temperatures (F1,111 = 120.012, p b 0.05) and a significant interaction between the two factors (F2,111 = 7.028, p b 0.05) . A significant interaction between temperature and the log of PAHs’ concentration (exposure levels) (F1,111 = 58.838, pb 0.05) was also found, while no significant interactions were found either between PAHs and the log of the concentration (F2,111 = 0.674, p >0.05) nor among temperature, PAHs and the log of the concentration (F2,111 =2.980, p >0.05). The R2 was 0.942. 4. Discussion All the PAHs tested significantly reduced the average specific growth rate of T. chuii at concentrations between 0.070 and 0.360 mg L − 1 at both temperatures, with phenanthrene inducing significant effects at lower concentrations than anthracene and naphthalene (Table S1 and Table 1). The concentrations of PAHs tested are considerably higher
Temperature
anthracene
R2 R2
= 0.942 = 0.953
R2 R2
= 0.926 = 0.918
R2 R2
= 0.900 = 0.973
probit
naphthalene
phenanthrene
logConc Fig. 1. T. chuii growth inhibition probit curves as a function of log concentration of the three tested PAHs, at both temperatures (20 and 25 °C). The R2 obtained for each temperature is also indicated.
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than those that have been reported for seawater where the values are in general in the pg L − 1 range; for example, anthracene at concentrations of 464.8 pg L − 1, phenanthrene at concentrations of 2259 pg L − 1 and naphthalene at concentrations of 1784 pg L − 1 were found in the Baltic Sea (Witt, 1995), while anthracene at concentrations of 67.6 pg L− 1 and phenanthrene at concentrations of 700 pg L − 1 were found in the Atlantic Ocean (Nizzetto et al., 2008). Even in the case of oil spills, water concentrations of individual PAHs in the vicinity of the spillage are in general in the ng L − 1 range (González et al., 2006; Neff and Stubblefield, 1995). Thus, even considering that PAHs may have additive toxic effects (Barata et al., 2005), these concentrations were selected to investigate if temperature was able to modify the toxicity of PAHs to marine microalgae and not to simulate ecologically relevant exposure scenarios. The IC50 values determined in the present study for anthracene, naphthalene and phenanthrene (Table 2) are in the range of corresponding values that have been reported for other microalgae species, despite the difficulty of direct comparisons due to the use of different study species and test conditions (e.g. light and other exposure conditions, saltwater versus freshwater used as test medium). For example, the 7-day IC50s of phenanthrene, naphthalene and anthracene to the freshwater microalgae Scenedesmus subspicatus at 25± 1 °C were found to be 50.24 mg L − 1 (95%CL: 34.88–72.50), 68.21 mg L− 1 (95%CL: 54.32–102.90) and 1.04 mg L − 1 (95% CL: 0.74–1.50), respectively (Djomo et al., 2004), while a 96 h IC50 of 0.347 mg L − 1 of phenanthrene to the marine microalgae Phaeodactylum tricornutum was determined at 22 ± 2 °C (Okay and Karacik, 2007). Based on the IC50s, the ranking order of toxicity of the PAHs tested to T. chuii at both 20 and 25 °C was phenanthrene > naphthalene>anthracene (Table 2). These findings are in good agreement with the PAHs toxicity ranking found for the marine microalgae Isochrysis galbana (fluoranthene ≥ pyrene > phenanthrene>naphthalene) by other authors (Pérez et al., 2010), but not with the ranking determined to the freshwater S. subspicatus (anthracene> phenanthrene > naphthalene) (Djomo et al., 2004). Despite the difficulty of comparing results obtained with marine and freshwater species, this is an interesting difference deserving further investigation. As Fig. 1 and Table 2 show, the 5 °C raise in temperature increased the toxicity (population growth inhibition) of the PAHs tested to T. chuii. Although such magnitude of temperature increase in open sea waters is not expected to occur in the next years (IPCC, 2000; Meehl et al., 2007), 25 °C already occur in temperate regions, in some shallow water systems such as estuaries and coastal lagoons at least in some periods of the year (e.g. summer in temperate regions). Water temperature variation of 5 °C is also a common event in these areas especially in years of draught conditions reducing rivers’ influx (Fink et al., 2004; Ilarri et al., 2011; Marques et al., 2007). In addition to increasing phenanthrene, naphthalene and anthracene toxicity, the increment of temperature seems to have also a different effect at low and higher PAHs’ concentrations with stronger effects at the highest concentrations tested (Fig. 1). These findings suggest that in marine ecosystems with moderate or high levels of pollution, microalgae are more vulnerable to temperature variation than in low contaminated ecosystems. 5. Conclusions The present study investigated the potential of temperature increase (20 to 25 °C) to modify the toxicity of three polycyclic aromatic hydrocarbons (anthracene, phenanthrene and naphthalene) to the marine planktonic microalgae T. chuii. The 5 °C raise of temperature significantly increased the toxicity of all the PAHs tested with stronger toxic effects at high PAHs’ exposure concentrations. These findings indicate that temperature affects the toxicity of chemical contaminants and suggest that in moderate or highly PAH polluted ecosystems marine microalgae are particularly vulnerable to temperature increase, highlighting the need of more research on the combined effects of temperature and chemical
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stress and of considering temperature variation on marine ecological risk assessments. Supplementary materials related to this article can be found online at doi:10.1016/j.seares.2012.02.004.
Acknowledgements We would like to thank Joana Rocha and Sofia Santos for the preliminary experiments on single PAH toxicity experiments done, and not included in the present study, and to Dr. Laura Guimarães for valuable discussions and help on the statistical analysis. This work was conducted in the scope of the RAMOCS project “Implementation of risk assessment methodologies for oil and chemical spills in the European main environment,” financially supported by the Portuguese Foundation for the Science and Technology (FCT) and FEDER funds (ERA-AMPERA/0001/2007) in the framework of the EU ERA-NET AMPERA (ERAC-CT2005-016165). Luis R. Vieira has a post-doc fellowship of FCT (SFRH/BPD/47407/2008) funded by national funds of the “MCTES – Ministério da Ciência, Tecnologia e do Ensino Superior” and of the European Social Fund. References Albaigés, J., Morales-Nin, B., Vilas, F., 2006. The prestige oil spill: a scientific response. Marine Pollution Bulletin 53, 205–207. Barata, C., Calbet, A., Saiz, E., Ortiz, L., Bayona, J.M., 2005. Predicting single and mixture toxicity of petrogenic polycyclic aromatic hydrocarbons to the copepod Oithona davisae. Environmental Toxicology and Chemistry 24, 2992–2999. Bellas, J., Saco-Álvarez, L., Nieto, O., Beiras, R., 2008. Ecotoxicological evaluation of polycyclic aromatic hydrocarbons using marine invertebrate embryo-larval bioassays. Marine Pollution Bulletin 57, 493–502. Crone, T.J., Tolstoy, M., 2010. Magnitude of the 2010 Gulf of Mexico oil leak. Science 330, 634. Djomo, J.E., Dauta, A., Ferrier, V., Narbonne, J.F., Monkiedje, A., Njine, T., Garrigues, P., 2004. Toxic effects of some major polyaromatic hydrocarbons found in crude oil and aquatic sediments on Scenedesmus subspicatus. Water Research 38, 1817–1821. Echeveste, P., Agustí, S., Dachs, J., 2011. Cell size dependence of additive versus synergetic effects of UV radiation and PAHs on oceanic phytoplankton. Environmental Pollution 159, 1307–1316. Fathalla, E.M., Andersson, J.T., 2011. Products of polycyclic aromatic sulfur heterocycles in oil spill photodegradation. Environmental Toxicology and Chemistry 30, 2004–2012. Ferreira, C., Nunes, B., Henriques-Almeida, J., Guilhermino, L., 2007. Acute toxicity of oxytetracycline and florfenicol to green unicellular algae Tetraselmis chuii and the hypersaline crustacean Artemia parthenogenetica. Ecotoxicology and Environmental Safety 67, 452–458. Fink, A.H., Brücher, T., Krüger, A., Leckebusch, G.C., Pinto, J.G., Ulbrich, U., 2004. The 2003 European summer heatwaves and drought—synoptic diagnosis and impacts. Weather 59, 209–216. Finney, D.J., 1971. Probit Analysis. Cambridge University Press, Cambridge. González, J.J., Viñas, L., Franco, M.A., Fumega, J., Soriano, J.A., Grueiro, G., Muniategui, S., López-Mahía, P., Prada, D., Bayona, J.M., Alzaga, R., Albaigés, J., 2006. Spatial and temporal distribution of dissolved/dispersed aromatic hydrocarbons in seawater in the area affected by the Prestige oil spill. Marine Pollution Bulletin 53, 250–259. Guillard, R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith, W.L., Chanley, M.H. (Eds.), Culture of Marine Invertebrate Animals. Plenum Press, New York, pp. 26–60. Guillard, R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Canadian Journal of Microbiology 8, 229–239. Ilarri, M.I., Antunes, C., Guilhermino, L., Sousa, R., 2011. Massive mortality of the Asian clam Corbicula fluminea in a highly invaded area. Biological Invasions 13, 277–280. IPCC, 2007. Climate Change 2007: Synthesis Report. In: Pachauri, R.K., Reisinger, A. (Eds.), Contribution of Working Groups I, II and III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. IPCC, Geneva, Switzerland. 104 pp. IPCC, 2000. In: Nakicenovic, N., Swart, R. (Eds.), Special Report on Emissions Scenarios. Cambridge University Press, UK. 570 pp. Leite, M.B.N.L., de Araújo, M.M.S., Nascimento, I.A., da Cruz, A.C.S., Pereira, S.A., do Nascimento, N.C., 2011. Toxicity of water-soluble fractions of biodiesel fuels derived from castor oil, palm oil, and waste cooking oil. Environmental Toxicology and Chemistry 30, 893–897. Lyons, B.P., Pascoe, C.K., McFadzen, I.R.B., 2002. Phototoxicity of pyrene and benzo[a] pyrene to embryo-larval stages of the pacific oyster Crassostrea gigas. Marine Environmental Research 54, 627–631. Marques, S.C., Azeiteiro, U.M., Martinho, F., Pardal, M.Â., 2007. Climate variability and planktonic communities: the effect of an extreme event (severe drought) in a southern European estuary. Estuarine, Coastal and Shelf Science 73, 725–734.
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Multiple stress effects on marine planktonic organisms: Influence of temperature on the toxicity of polycyclic aromatic hydrocarbons to Tetraselmis chuii L.R. Vieira ⁎, L. Guilhermino CIIMAR—Interdisciplinary Centre of Marine and Environmental Research, Laboratory of Ecotoxicology and Ecology, University of Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal ICBAS—Institute of Biomedical Sciences of Abel Salazar, University of Porto, Department of Populations Studies, Laboratory of Ecotoxicology, Largo Prof. Abel Salazar, 4099-003 Porto, Portugal
a r t i c l e
i n f o
Article history: Received 29 May 2011 Received in revised form 6 February 2012 Accepted 7 February 2012 Available online 15 February 2012 Keywords: Plankton Global Warming PAHs Oil Spills Multiple Stressors
a b s t r a c t In the present context of global warming and increasing long-range transport of oil and goods by sea potentially resulting in oil spills, more knowledge on the toxicological interactions between temperature and oil components on marine organisms is urgently needed. Therefore, the effects of temperature increase on the toxicity of three polycyclic aromatic hydrocarbons (PAH; anthracene, phenanthrene and naphthalene) to the marine planktonic algae Tetraselmis chuii were investigated under laboratory conditions. T. chuii cultures were exposed for 96 h to different concentrations of each of the test substances at both 20 and 25 °C. Effect criterion was the inhibition of culture growth assessed at 24 h intervals. All the PAHs significantly reduced T. chuii growth after 96 h of exposure with 20% inhibition concentrations between 0.052 and 1.124 mg L− 1 at 20 °C, and between 0.048 and 0.831 mg L− 1 at 25 °C. At both temperatures, the ranking, in order of decreasing toxicity based on the 50% inhibition concentration, was phenanthrene > naphthalene > anthracene. The increase of temperature by 5 °C significantly increased the toxicity of all the PAHs tested. These findings highlight the importance of considering temperature variation in the ecological risk assessment of oil and other chemical spills in the marine environment, and the need of more research on the toxic effects of multiple stressors on marine organisms. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Recent oil spills in the marine environment showed again how negative the immediate ecological and economic consequences of these events can be (Albaigés et al., 2006; Crone and Tolstoy, 2010). Long term studies on the effects of oils spill events that occurred several years ago have shown that environmental conditions play a determinant role on the degradation and bioavailability of oil components and on their toxicity to marine organisms (Fathalla and Andersson, 2011; Neff et al., 2011; Tansel et al., 2011). These findings highlight the need of improving prevention systems and ecological risk assessment procedures, and of getting more knowledge on the ecological status of vulnerable areas to be protected, on both short and long term toxic effects of oil components, and on the effects of multiple stressors on marine organisms. The interactions between temperature and oil components are of special relevance considering the present context of global warming (IPCC, 2007) and the increasing trend of long-range transport of oils and goods by sea
⁎ Corresponding author at: CIIMAR—Centro Interdisciplinar de Investigação Marinha e Ambiental, Laboratório de Ecotoxicologia e Ecologia, Rua dos Bragas, 289, 4050-123 Porto, Portugal. Tel.: + 351 223401800. E-mail address: [email protected] (L.R. Vieira). 1385-1101/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.seares.2012.02.004
which potentially raises the risk of oil spills despite the improvement of preventive regulation and measures (Martínez-Gómez et al., 2009). Among oil components, polycyclic aromatic hydrocarbons (PAHs) are particularly important from an ecotoxicological point of view due to their toxicity, since several of them or their metabolites have been found to cause genotoxic, carcinogenic, endocrine disruption and other toxic effects in marine organisms (e.g. Thomas, 1988; Wessel et al., 2010; Zheng et al., 2005, 2011). The toxicity of oils and PAHs to marine organisms has been shown to be increased by temperature (Saeed et al., 1998) and UV radiation (Bellas et al., 2008; Echeveste et al., 2011; Lyons et al., 2002; Meng et al., 2007), among other factors. In marine ecosystems, planktonic algae populations have key roles as primary producers (hereafter indicated as producers). Since they are planktonic organisms they may be particularly exposed to oils immediately after spillages of these products, to oil spots floating at the sea surface for variable periods of time, and to inputs from other sources, including continental ones. Thus, the objective of the present study was to investigate the effects of temperature increase (from 20 to 25 °C) on the toxicity of three polycyclic aromatic hydrocarbons (anthracene, naphthalene and phenanthrene) to the marine planktonic algae Tetraselmis chuii in laboratory conditions. T. chuii was selected for this study because it has been used as a representative of marine phytoplankton in previous ecotoxicological studies (e.g. Ferreira et al., 2007; Leite et al., 2011; Nunes et al., 2005; Sibila et
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al., 2008) and it is a native species in marine ecosystems of temperate regions. The inhibition of algae culture growth was used as effect criterion as recommended in international protocols (e.g. OECD, 2006; USEPA, 2002) to access the toxic effects of chemical substances on microalgae.
2.4. Data analysis Following the OECD guideline 201 (OECD, 2006), the T. chuii average specific growth rates (μ) per day were calculated as the logarithmic increase of biomass in each test recipient, over the entire test duration (4 days), according to the equation:
2. Materials and methods 2.1. Chemicals Tested chemicals were phenanthrene (CAS no. 85-01-8), purchased from Fluka GmbH (Buchs, Switzerland), anthracene (CAS no. 120-12-7) and naphthalene (CAS no. 91-20-3), both purchased from Sigma– Aldrich Chemical (Steinheim, Germany). All the other chemicals were acquired from Sigma–Aldrich Chemical (Steinheim, Germany). 2.2. Algae culture conditions The green unicellular marine algae T. chuii was kindly provided by the Instituto de Ciencias Marinas de Andalucía (CSIC), Spain. This species was cultured in the laboratory under standardized abiotic conditions (24 h of solar spectrum light at 20 ± 1 °C), generally following the recommendations of OECD (2006). Test medium was f/2 Guillard's medium (Guillard, 1975; Guillard and Ryther, 1962), sterilized by autoclaving (Uniclave 88 - AJC, Portugal) at 121 °C for 35 min. All cultures were routinely monitored by microscopic observation (Leica DM 2000) to assure the normal status and growth of T. chuii. 2.3. Toxicity tests Tests were carried out for 96 h in static conditions, following the OECD guideline 201 “Alga, Growth Inhibition Test” (OECD, 2006). For each tested substance, two independent tests were carried out: one at 20 °C and the other at 25 °C, in temperature controlled rooms under 24 h of light (solar spectrum lamps (Arcadia, model FO30), 90 μE/m 2/s). The distance between the source of light and test recipients was 14 cm in all treatments and tests; test recipients were never protected from light during the exposure period to simulate environmental conditions; thus, the toxicity assessed corresponds to the overall effects of parental compounds, metabolites and environmental degradation products formed during the exposure period. Individual stock solutions (500 mg L − 1) of anthracene, phenanthrene and naphthalene were prepared in acetone (100%). For each PAH, test solutions were obtained by dilution of the corresponding stock solution in f/2 medium. The nominal test concentrations were 0.09, 0.18, 0.36, 0.72, 1.44, 2.88 and 5.76 mg L − 1 for anthracene; 0.045, 0.09, 0.18, 0.36, 0.72, 1.44 and 2.88 mg L − 1 for naphthalene and 0.035, 0.07, 0.14, 0.28, 0.56, 1.12 and 2.24 mg L − 1 for phenanthrene. In each test and for each temperature, two controls were included in the experimental design: one with f/2 medium only (hereafter designated as “control”) and another one (hereafter indicated as “acetone-control”) with the highest concentration of acetone used in the corresponding PAH treatments, namely 11.52, 5.76 and 4.48 mL of acetone per L of test medium for anthracene, naphthalene and phenanthrene, respectively. Test recipients (500 mL Erlenmeyer flasks) were filled with 400 mL of filtered culture medium, with perforated rubber stoppers, equipped with aeration systems and sterilized (Uniclave 88 - AJC, Portugal) at 121 °C for 35 min prior to algae inoculation. Treatments were inoculated with a volume of exponential growing algal cultures to obtain the concentration of 1 × 104 cells mL− 1 in test medium at the beginning of the assays. In each assay, three replicates per treatment were used. Growth was determined by cell counts of samples taken from each treatment at 0, 24, 48, 72 and 96 h, using a Neubauer Improved bright-line chamber and a microscope (Leica DM 2000), after cell fixation by Lugol. During the test, medium temperature and pH were monitored every 24 h (pH meter 3310 Jenway).
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μ ij ¼
lnX j lnX i 1 day tj ti
where μi-j is the average specific growth rate from time i to j, Xi is the biomass at time i and Xj is the biomass at time j. To assess the effects of each PAH on the algae growth at each temperature and to determine the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC), different treatments were compared by one-way ANOVA, followed by Dunnett's test comparing each PAH treatment and the acetone-control against the control (Zar, 1996). Prior to ANOVA, data were tested for normality of distribution (Kolmogorov– Smirnov normality test) and homogeneity of variance (Levine's test) (Zar, 1996) and data transformations were made when necessary. For each PAH and temperature, the percent inhibition of growth rate was calculated for each treatment replicate using the equation (OECD, 2006): %I r ¼
μ c −μ t 100 μc
where %Ir is the percent inhibition in average specific growth rate; μC is the mean value for average specific growth rate (μ) in the control group since no significant differences in the average specific growth rates were found between the control and the acetone-control in any of the toxicity tests (Table S1); and μt is the average specific growth rate for the treatment replicate. The %Ir values calculated for each temperature, PAH and test replicate were transformed by probit analysis (Finney, 1971) and plotted against the logarithm of the test substance concentration to obtain the toxicity curves. From each, the 20% and 50% inhibition concentrations (IC20 and IC50, respectively) were calculated. To compare the effects of temperature on the toxicity of different PAHs, all the toxicity curves were compared using a two-way analysis of covariance (ANCOVA) (Zar, 1996) with the different exposure levels as covariates, and PAHs and temperatures as fixed factors in a full-factorial model. All statistical analyses were performed using the SPSS 17.0 software package and differences were considered statistically significant when p b 0.05. 3. Results The microalgae average specific growth rates over the period of the test (4 days), for each tested PAH, at both temperatures are provided in Supplementary Data (see Table S1). In all the assays, significant differences in average specific growth rates among differences were found at both 20 °C (anthracene: F(8,26) = 229.9, p b 0.05; naphthalene: F(8,26) = 113.5, p b 0.05; phenanthrene: F(8,26) = 145.8, Table 1 Comparison of the no observed effect concentrations (NOECs) and the lowest observed effect concentrations (LOECs) of anthracene, naphthalene and phenanthrene to T. chuii at 20 and 25 °C. 20 °C
−1
Anthracene (mg L ) Naphthalene (mg L− 1) Phenanthrene (mg L− 1)
25 °C
NOEC
LOEC
NOEC
LOEC
0.180 0.180 0.035
0.360 0.360 0.070
0.090 0.180 0.035
0.180 0.360 0.070
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Table 2 Estimated 20% and 50% growth inhibition concentrations (IC20s and IC50s, respectively) of anthracene, naphthalene and phenanthrene to T. chuii after 96 h of exposure at 20 and 25 °C. 95% confidence intervals are indicated within brackets. 20 °C
−1
Anthracene (mg L ) Naphthalene (mg L− 1) Phenanthrene (mg L− 1)
25 °C
IC20 (95%CI)
IC50 (95%CI)
IC20 (95%CI)
IC50 (95%CI)
1.124 (0.570–1.711) 0.767 (0.562–0.964) 0.052 (0.038–0.067)
3.326 (2.718–4.233) 1.813 (1.571–2.136) 1.316 (1.032–1.729)
0.831 (0.388–1.207) 0.465 (0.255–0.656) 0.048 (0.033–0.065)
2.145 (1.734–2.743) 0.992 (0.788–1.301) 0.262 (0.236–0.291)
p b 0.05) and 25 °C (anthracene: F(8,26) = 326.1, p b 0.05; naphthalene: F(8,26) = 270.5, p b 0.05; phenanthrene: F(8,26) = 447.8, p b 0.05). As indicated by the Dunnett's tests carried out for each individual PAH and temperature, no significant differences between the control and the acetone-control were found, the NOECs determined for the average specific growth rates varied from 0.035 mg L − 1 and 0.180 mg L − 1, while LOECs ranged from 0.070 mg L − 1 to 0.360 mg L − 1 for both temperatures (Table 1). The IC20 and IC50 values calculated from the toxicity curves (log concentration vs. probit transformation of %Ir values) are shown in Table 2. At 20 °C, IC20s ranged from 0.052 for phenanthrene to 1.124 mg L − 1 for anthracene, while the corresponding values at 25 °C ranged from 0.048 to 0.831 mg L − 1 for phenanthrene and anthracene, respectively. At 20 °C, the IC50s ranged from 1.316 mg L − 1 for phenanthrene to 3.326 mg L − 1 for anthracene, while at 25 °C they ranged from 0.262 for phenanthrene to 2.145 mg L − 1 for anthracene. The toxicity curves (log of PAH concentration vs. % of growth inhibition) obtained for different PAHs at both 20 and 25 °C are
shown in Fig. 1. Significant differences among the toxicity curves were found (ANCOVA: F11,111 =165.012, p b 0.05, observed power= 1.0), with statistical significant differences among PAHs (F2,111 = 109.117, p b 0.05), between temperatures (F1,111 = 120.012, p b 0.05) and a significant interaction between the two factors (F2,111 = 7.028, p b 0.05) . A significant interaction between temperature and the log of PAHs’ concentration (exposure levels) (F1,111 = 58.838, pb 0.05) was also found, while no significant interactions were found either between PAHs and the log of the concentration (F2,111 = 0.674, p >0.05) nor among temperature, PAHs and the log of the concentration (F2,111 =2.980, p >0.05). The R2 was 0.942. 4. Discussion All the PAHs tested significantly reduced the average specific growth rate of T. chuii at concentrations between 0.070 and 0.360 mg L − 1 at both temperatures, with phenanthrene inducing significant effects at lower concentrations than anthracene and naphthalene (Table S1 and Table 1). The concentrations of PAHs tested are considerably higher
Temperature
anthracene
R2 R2
= 0.942 = 0.953
R2 R2
= 0.926 = 0.918
R2 R2
= 0.900 = 0.973
probit
naphthalene
phenanthrene
logConc Fig. 1. T. chuii growth inhibition probit curves as a function of log concentration of the three tested PAHs, at both temperatures (20 and 25 °C). The R2 obtained for each temperature is also indicated.
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than those that have been reported for seawater where the values are in general in the pg L − 1 range; for example, anthracene at concentrations of 464.8 pg L − 1, phenanthrene at concentrations of 2259 pg L − 1 and naphthalene at concentrations of 1784 pg L − 1 were found in the Baltic Sea (Witt, 1995), while anthracene at concentrations of 67.6 pg L− 1 and phenanthrene at concentrations of 700 pg L − 1 were found in the Atlantic Ocean (Nizzetto et al., 2008). Even in the case of oil spills, water concentrations of individual PAHs in the vicinity of the spillage are in general in the ng L − 1 range (González et al., 2006; Neff and Stubblefield, 1995). Thus, even considering that PAHs may have additive toxic effects (Barata et al., 2005), these concentrations were selected to investigate if temperature was able to modify the toxicity of PAHs to marine microalgae and not to simulate ecologically relevant exposure scenarios. The IC50 values determined in the present study for anthracene, naphthalene and phenanthrene (Table 2) are in the range of corresponding values that have been reported for other microalgae species, despite the difficulty of direct comparisons due to the use of different study species and test conditions (e.g. light and other exposure conditions, saltwater versus freshwater used as test medium). For example, the 7-day IC50s of phenanthrene, naphthalene and anthracene to the freshwater microalgae Scenedesmus subspicatus at 25± 1 °C were found to be 50.24 mg L − 1 (95%CL: 34.88–72.50), 68.21 mg L− 1 (95%CL: 54.32–102.90) and 1.04 mg L − 1 (95% CL: 0.74–1.50), respectively (Djomo et al., 2004), while a 96 h IC50 of 0.347 mg L − 1 of phenanthrene to the marine microalgae Phaeodactylum tricornutum was determined at 22 ± 2 °C (Okay and Karacik, 2007). Based on the IC50s, the ranking order of toxicity of the PAHs tested to T. chuii at both 20 and 25 °C was phenanthrene > naphthalene>anthracene (Table 2). These findings are in good agreement with the PAHs toxicity ranking found for the marine microalgae Isochrysis galbana (fluoranthene ≥ pyrene > phenanthrene>naphthalene) by other authors (Pérez et al., 2010), but not with the ranking determined to the freshwater S. subspicatus (anthracene> phenanthrene > naphthalene) (Djomo et al., 2004). Despite the difficulty of comparing results obtained with marine and freshwater species, this is an interesting difference deserving further investigation. As Fig. 1 and Table 2 show, the 5 °C raise in temperature increased the toxicity (population growth inhibition) of the PAHs tested to T. chuii. Although such magnitude of temperature increase in open sea waters is not expected to occur in the next years (IPCC, 2000; Meehl et al., 2007), 25 °C already occur in temperate regions, in some shallow water systems such as estuaries and coastal lagoons at least in some periods of the year (e.g. summer in temperate regions). Water temperature variation of 5 °C is also a common event in these areas especially in years of draught conditions reducing rivers’ influx (Fink et al., 2004; Ilarri et al., 2011; Marques et al., 2007). In addition to increasing phenanthrene, naphthalene and anthracene toxicity, the increment of temperature seems to have also a different effect at low and higher PAHs’ concentrations with stronger effects at the highest concentrations tested (Fig. 1). These findings suggest that in marine ecosystems with moderate or high levels of pollution, microalgae are more vulnerable to temperature variation than in low contaminated ecosystems. 5. Conclusions The present study investigated the potential of temperature increase (20 to 25 °C) to modify the toxicity of three polycyclic aromatic hydrocarbons (anthracene, phenanthrene and naphthalene) to the marine planktonic microalgae T. chuii. The 5 °C raise of temperature significantly increased the toxicity of all the PAHs tested with stronger toxic effects at high PAHs’ exposure concentrations. These findings indicate that temperature affects the toxicity of chemical contaminants and suggest that in moderate or highly PAH polluted ecosystems marine microalgae are particularly vulnerable to temperature increase, highlighting the need of more research on the combined effects of temperature and chemical
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stress and of considering temperature variation on marine ecological risk assessments. Supplementary materials related to this article can be found online at doi:10.1016/j.seares.2012.02.004.
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