Development and application of an oil toxicity and exposure model,OilToxEx

An oil toxicity and exposure model (OilToxEx) was developed and validated for estimation of impacts to aquatic organisms resulting from acute exposure to spilled oil. Because oil exposure is shorter than the time required for equilibrium between the organism and the water to be reached, the time and temperature dependence of toxicity is addressed. Oil toxicity is a function of aromatic composition and the toxicity of individual aromatics in the mixture. Lethal concentration to 50% of exposed organisms (LC50), as a function of octanol-water partition coefficient (Kow), and an additive model are used to estimate the toxicity of monoaromatic and polycyclic aromatic hydrocarbon mixtures in water-soluble fractions (WSF) and oil-in-water dispersions (OWD) of oil. The toxicity model was verified by comparison with oil bioassay data where the exposure concentrations of aromatics were measured. The observed toxicity in the bioassays could be accounted for by the additive narcotic effects of the dissolved aromatics in the exposure media. Predicted LC50s were compared to those calculated from measured concentrations after spills to verify the exposure model for field conditions. These results indicate that the additive toxicity and exposure model may be used to estimate toxicity of untested oils and spill conditions.     

Uptake and elimination of hydrophobic organic contaminants in estuarine copepods: an experimental study

Polycyclic aromatic hydrocarbons (PAHs) are considered to be rapidly biotransformed by organisms, whereas polychlorinated biphenyls (PCBs) are strongly bioaccumulated. In the present study, the estuarine copepod Eurytemora affinis was exposed in a continuous flow-through system to dissolved PAH (500 ng/L) and PCB (300 ng/L) mixtures for 86 h, whereas control groups were placed in a continuous flow-through system with clean water. Both PCB and PAH body residues were measured and compared in exposed and in nonexposed copepods to assess the uptake and the elimination of these two contaminant classes in this copepod species. After the exposure, exposed copepods exhibited concentration factors, based on a dry-weight basis, of 25, 750, and 1,200, respectively, for total PCBs and PAHs. The lower concentrations of PAHs in the nonexposed versus exposed copepods in contrast to small differences for PCBs suggest a higher rate of metabolism of PAHs compared with PCBs and could explain the differences observed in the accumulation. Furthermore, uptake as well as elimination of both PCBs and PAHs were compound selective in E. affinis. Therefore, higher-molecular-weight PCBs and PAHs were preferentially accumulated, while lower-molecular-weight compounds were preferentially eliminated. These results suggest the importance of copepods in the biogeochemical cycles of hydrophobic organic contaminants in estuarine ecosystems.     

Distribution of polycyclic aromatic hydrocarbons in soils and terrestrial biota after a spill of crude oil in Trecate,Italy

Ecological and human health exposures from soil-based petroleum-derived contaminants commonly are estimated by using soil-to-biota transfer factors that usually are based on octanol-water partitioning. Few studies of biota have been conducted in relation to spills of crude oils in terrestrial environments. After a large blowout of crude oil in northern Italy in 1994, the distribution of polycyclic aromatic hydrocarbons (PAHs) was examined over time and space in soils, uncultivated wild vegetation, insects, mice, and frogs in the area. Within two years of the blowout, PAH concentrations declined to background levels over much of the area where initial concentrations were within an order of magnitude above background, but had not declined to background in areas where starting concentrations exceeded background by two orders of magnitude. Octanol-water partitioning and extent of alkylation explained much of the variance in uptake of PAHs by plants and animals. The PAHs with lower octanol-water partition coefficients (K(ow)s) and higher-alkylated PAHs had higher biota-soil accumulation factors (BSAFs) than did high-K(ow) and unalkylated forms. The BSAFs for PAHs with higher K(ow)s were very low for plants, but much higher for animals, with frogs accumulating more of these compounds than other species.     

Accumulation and elimination of 16 polycyclic aromatic compounds in the earthworm (Eisenia fetida)

Accumulation and elimination of different polycyclic aromatic compounds (PACs) were studied in earthworms (Eisenia fetida) exposed to contaminated soil from an old gasworks site. In total, 12 polycyclic aromatic hydrocarbons (PAHs), two N- and S-heterocyclic PACs, and two PAC-quinones were included in the study. Peak-shaped accumulation curves were found for many of the compounds. After 19 d of exposure, the ratio between concentrations in worm lipids and soil organic matter was 0.02 on average. The half-lives of the PACs were relatively long, between 2 and 11 d. The elimination rate constants, k2, correlated both with literature-derived octanol-water partition coefficients (Kow) for PAHs (r2 = 0.93) and the computed polarizability (r2 = 0.88) of all the compounds. The elimination rate constants of PAHs are comparable to those of PCBs found in earlier studies, and the linear regression coefficient, r2, of k2 against Kow for PAHs and PCBs together was 0.93.     

Aryl hydrocarbon receptor-independent toxicity of weathered crude oil during fish development

Polycyclic aromatic hydrocarbons (PAHs), derived largely from fossil fuels and their combustion, are pervasive contaminants in rivers, lakes, and nearshore marine habitats. Studies after the Exxon Valdez oil spill demonstrated that fish embryos exposed to low levels of PAHs in weathered crude oil develop a syndrome of edema and craniofacial and body axis defects. Although mechanisms leading to these defects are poorly understood, it is widely held that PAH toxicity is linked to aryl hydrocarbon receptor (AhR) binding and cytochrome P450 1A (CYP1A) induction. Using zebrafish embryos, we show that the weathered crude oil syndrome is distinct from the well-characterized AhR-dependent effects of dioxin toxicity. Blockade of AhR pathway components with antisense morpholino oligonucleotides demonstrated that the key developmental defects induced by weathered crude oil exposure are mediated by low-molecular-weight tricyclic PAHs through AhR-independent disruption of cardiovascular function and morphogenesis. These findings have multiple implications for the assessment of PAH impacts on coastal habitats.     

Predicting bioavailability of PAHs and PCBs with porewater concentrations measured by solid-phase microextraction fibers

Bioaccumulation of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) was measured in the deposit-feeding oligochaete Ilyodrilus templetoni exposed for 28?d to Anacostia River sediment (Washington, DC, USA) and to an initially uncontaminated sediment from Brown Lake (Vicksburg, MS, USA) sequentially diluted with 3 to 25% contaminated New Bedford Harbor sediment (New Bedford, MA, USA). The Anacostia River sediment studies represented exposure to a historically contaminated sediment with limited availability, whereas exposure to the other sediment included both the historically contaminated New Bedford Harbor sediment and fresh redistribution of contaminants into the Brown Lake sediments. Organism tissue concentrations did not correlate with bulk sediment concentrations in the Anacostia River sediment but did correlate with the sequentially diluted sediment. Porewater concentrations measured via disposable solid-phase microextraction fiber (SPME) with polydimethylsiloxane (PDMS), however, correlated well with organism uptake in all sediments. Bioaccumulation was predicted well by a linear relationship with the product of porewater concentration and compound octanol-water partition coefficient (Anacostia, slope?=?1.08, r2?=?0.76; sequentially diluted sediments, slope = 1.24, r2?=?0.76). The data demonstrate that the octanol-water partition coefficient is a good indicator of the lipid-water partition coefficient and that porewater concentrations provide a more reliable indicator of bioaccumulation in the organism than sediment concentrations, even when the route of uptake is expected to be via sediment ingestion.     

Modeling polycyclic aromatic hydrocarbon bioaccumulation and metabolism in time-variable early life-stage exposures

Recent laboratory investigations into the bioaccumulation and toxicity of polycyclic aromatic hydrocarbons (PAH) have focused on low-level, time-variable exposures to early life-stage fish. Polycyclic aromatic hydrocarbon body-burden residues reported in these studies were lower than critical body-burden residues predicted by the target lipid model (TLM). To understand this discrepancy, a time-variable uptake and depuration model of PAH bioaccumulation was developed. Kinetic constants were fit using measured exposure and tissue concentrations. The resulting lipid-water partition coefficients (K(LW)) were uncorrelated with the octanol-water partition coefficient (K(OW))--a qualitatively unrealistic finding considering that numerous studies have reported a positive correlation between the two. Because PAHs are known to be metabolized, the comparison of K(LW) with K(OW) suggests that metabolism may be occurring in early life-stage fish. Therefore, the uptake and depuration model was modified to include metabolism while assuming linearity of K(LW) with K(OW). Calculated metabolism rates were positively correlated with K(OW)--a finding qualitatively similar to those of other studies. The present study provides a reasonable explanation for the discrepancy between the TLM predictions and the measured toxic effect levels. Given the time-variable exposure concentrations, the maximum measured body burdens used to relate to toxic effects may be underestimated. In addition, the maximum body burden of parent PAH plus metabolites may be a better measure in relating tissue concentrations to toxic effects. Incorporating these refinements in relating body burdens to toxic effects may result in a better comparison between TLM predictions and measured effect levels.     

Short-term ecological risks of depositing contaminated sediment on arable soil

Sediments act as sinks of suspended material from surface water. Dredging of regional waters and subsequent disposal of the sediment on soil may lead to contamination of the soil, in some cases resulting in exceedance of soil quality standards. Soil quality standards are based on total concentrations. Total levels, however, do not always give an indication of adverse effects in soil ecosystems. Instead, truly bioavailable concentrations should be used as indicators. In this study we aim to test a set of suited indicators. We carried out partition and accumulation assays with metals and polycyclic aromatic hydrocarbons (PAHs) in soils and mixtures of soil and sediment, as well as a limited number of toxicity bioassays. We also investigated the rate of disappearance of PAHs from mixtures of sediments and soils. The experiments confirm that total levels indeed are not indicative of truly occurring toxic effects: mixing of highly contaminated sediments with soil hardly gave rise to either additional accumulation of metals and PAHs or excess toxicity. This indicates that the bioavailability of the metals and PAHs present in the sediment is limited. This general finding is confirmed by the low rate of disappearance of PAHs from the mixtures. It is concluded that inclusion of the aspects of bioavailability, mixture toxicity, and degradation, in the way described in this report, will solve the major limitations of the current methodology of classification of contaminated sediments.     

Weathering and toxicity of marine sediments contaminated with oils and polycyclic aromatic hydrocarbons

Many sediments are contaminated with mixtures of oil residues and polycyclic aromatic hydrocarbons (PAHs), but little is known about the toxicity of such mixtures to sediment-dwelling organisms and the change in toxicity on weathering. In the present study, we investigated the effects of a seminatural, two-year weathering period on PAH/oil chemistry and toxicity in a marine sediment that had been spiked with three different oils (a gas oil, a lubricating oil, and a crude oil; all tested at five concentrations). Toxicity of bioavailable, pore water-accommodated oil/PAH fractions was quantified using a bacterial (Vibrio fischeri) assay and the in vitro chemical-activated luciferase expression assay (DR-CALUX; using conditions to detect PAHs). Results of chemical analyses pointed to (microbial) degradation of all three oils: Sediment oxygen demand during weathering increased with increasing oil concentration, total oil concentrations decreased to between 17 and 29% of initial levels, and resolved n-alkanes were depleted in weathered oil fractions. Furthermore, a shift in the relative importance of different boiling-point fraction ranges of the oils was observed on weathering. Generally, the lowest fraction range (C10-C16) disappeared, whereas the relative proportion of the highest (C28-C40) fraction range increased considerably. Remarkably, for the gas oil, this fraction shift was dependent on the oil concentration in sediment. Similarly, degradation of PAHs was strongly affected by the sedimentary oil content, indicating that the presence of oil stimulated PAH degradation. This phenomenon applied to both low- and high-molecular-weight PAHs, although the first group (3- and 4-ring PAHs) was degraded most. Results from the V. fischeri and DR-CALUX assay showed that in most cases, pore-water toxicity decreased on weathering. Combining the assay responses with chemical data indicated that the observed toxicity probably was not caused by the analyzed PAHs but, rather, by specific oil constituents instead.     

Effects of Dispersant and Oil on Survival and Swimming Activity in a Marine Copepod

Knowledge of lethal and sublethal effects of crude oil and dispersants on mesozooplankton are important to understanding ecosystem impacts of oil spills in marine environments. Here we (1) establish median lethal concentrations for water accommodated fractions of Corexit EC9500A dispersant, MC-252 crude oil (WAF), and dispersed crude oil (CEWAF) for the coastal copepod Labidocera aestiva, and (2) assess acute effects on L. aestiva swimming activity. Mortality assays with L. aestiva support that copepods are more sensitive than other zooplankton taxa to dispersant toxicity, while WAF and CEWAF are generally similar in their toxicity to this copepod species and other zooplankton. Acute effects on L. aestiva activity included impaired swimming upon WAF and CEWAF exposure. These results highlight that copepods are particularly sensitive to dispersant exposure, with acute effects on survival most evident with dispersant alone, and on swimming behavior when dispersant is mixed with crude oil.     

Bioaccumulation of polycyclic aromatic compounds: 2. Modeling bioaccumulation in marine organisms chronically exposed to dispersed oil

Within the frame of a large environmental study, we report on a research program that investigated the potential for bioaccumulation and subsequent effect responses in several marine organisms exposed to chronic levels of dispersed crude oil. Body burden can be estimated from kinetic parameters (rate constants for uptake and elimination), and appropriate body burden-effect relationships may improve assessments of environmental risks or the potential for such outcomes following chronic discharges at sea. We conducted a series of experiments in a flow-through system to describe the bioaccumulation kinetics of polycyclic aromatic hydrocarbons (PAH) at low concentrations of dispersed crude oils. Mussels (Mytilus edulis) and juvenile turbot (Scophthalmus maximus) were exposed for periods ranging from 8 to 21 d. Postexposure, the organisms were kept for a period of 9 to 10 d in running seawater to study elimination processes. Rate constants of uptake (k1) and elimination (k2) of the PAHs during and following exposure were calculated using a first-order kinetic model that assumed a decrease of the substances in the environment over time. The estimated bioconcentration factor was calculated from the ratio of k1/k2. The kinetic parameters of two-, three-, and four-ring PAHs in mussel and fish are compared with estimates based on hydrophobicity alone, expressed by the octanol-water partition coefficient, Kow (partitioning theory). A combination of reduced bioavailability of PAHs from oil droplets and degradation processes of PAHs in body tissues seems to explain discrepancies between kinetic rates based on Kow and actual kinetic rates measured in fish. Mussels showed a pattern more in compliance with the partitioning theory.     

Predicting the toxicity of neat and weathered crude oil: toxic potential and the toxicity of saturated mixtures

The toxicity of oils can be understood using the concept of toxic potential, or the toxicity of each individual component of the oil at the water solubility of that component. Using the target lipid model to describe the toxicity and the observed relationship of the solubility of oil components to log (Kow), it is demonstrated that components with lower log (Kow) have greater toxic potential than those with higher log (Kow). Weathering removes the lower-log (Kow) chemicals with greater toxic potential, leaving the higher-log (Kow) chemicals with lower toxic potential. The replacement of more toxically potent compounds with less toxically potent compounds lowers the toxicity of the aqueous phase in equilibrium with the oil. Observations confirm that weathering lowers the toxicity of oil. The idea that weathering increases toxicity is based on the erroneous use of the total petroleum hydrocarbons or the total polycyclic aromatic hydrocarbons (PAHs) concentration as if either were a single chemical that can be used to gauge the toxicity of a mixture, regardless of its makeup. The toxicity of the individual PAHs that comprise the mixture varies. Converting the concentrations to toxic units (TUs) normalizes the differences in toxicity. A concentration of one TU resulting from the PAHs in the mixture implies toxicity regardless of the specific PAHs that are present. However, it is impossible to judge whether 1 microg/L of total PAHs is toxic without knowing the PAHs in the mixture. The use of toxic potential and TUs eliminates this confusion, puts the chemicals on the same footing, and allows an intuitive understanding of the effects of weathering.     

Polycyclic aromatic hydrocarbon levels in mussels from Prince William Sound, Alaska, USA, document the return to baseline conditions

Bioavailable hydrocarbons in the Exxon Valdez oil spill zone in Prince William Sound (PWS; AK, USA) shorelines were at or near background levels in 2002, as indicated by low concentrations of polycyclic aromatic hydrocarbons (PAHs) in mussels (Mytilus trossulus) collected from sites throughout PWS. Total PAH (TPAH) minus parent naphthalene concentrations in mussels collected in 1998 to 2002 from sites oiled in 1989 were at or near reference-site values. Both oiled and reference sites included locations associated with past human and industrial activity (HA). Inclusion of the unoiled HA sites in the range of reference sites that define prespill conditions is consistent with federal regulations. For the period from 1998 to 2002, the geometric mean of TPAH concentrations for 218 mussel samples collected from 72 sites, including four HA sites that had been heavily oiled in 1989, is 54 ng/g dry weight (range, 2-1,190 ng/g). The maximum mussel TPAH concentrations are equivalent to a weathered-oil exposure dose to intertidal foragers that is one to three orders of magnitude less than the doses shown to cause sublethal effects in surrogate species. The geometric mean of TPAH concentrations for mussel samples from 28 locations not oiled in 1989 and unaffected by human use (NHA sites) is 28 ng/g (range, 3-355 ng/g), whereas the geometric mean of TPAH concentrations for mussel samples from 14 locations not oiled in 1989 and affected by human use (HA sites) is 106 ng/g (range, 2-12,056 ng/g). The range of data for the unoiled HA and NHA sites defines the background of bioavailable PAHs to mussels on western PWS shorelines that would have prevailed if the oil spill had not occurred. The low PAH concentrations in mussels from sites known to have subsurface oil residues demonstrates the low bioavailability of these spill remnants and, thus, are a low additional risk to foraging wildlife. The present study shows continuous exposure from four- to six-ring PAHs originating at HA sites in western PWS. At low concentrations, these PAHs are known to cause adverse biological effects. However, in the context of PWS, oiled and HA sites represent a small percentage (approximately 0.1-0.2%) of the total PWS shoreline.     

Mechanisms affecting the dissolution of nonaqueous phase liquids into the aqueous phase in slow-stirring batch systems

Understanding the kinetics of the exchange processes between nonaqueous phase liquids (NAPLs) and water is important in predicting the fate of anthropogenic compounds such as petroleum hydrocarbons, i.e., benzene, toluene, ethylbenzene, and xylene (BTEX) as well as polynuclear aromatic hydrocarbons (PAHs). Exchange processes occurring in the environment resemble the experimental setup of the slow-stirring method (SSM) designed to determine solubilities and octanol-water partition coefficients. Data obtained from SSM experiments for diesel fuel compounds are interpreted by a linear transfer model that is characterized by an aqueous molecular boundary layer and the water/NAPL equilibrium partition coefficient. For the chosen experimental setup, the boundary layer thickness is 2.42 x 10(-2) cm. Typical equilibration times lie between 1 and 2 d. Due to the temperature dependence of the aqueous diffusivity, this time increases with decreasing temperature. Transport within the NAPL phase can slow down the exchange process for the more water-soluble compounds (e.g., benzene) provided that the stirring rate exceeds a critical value.     

Photo-induced toxicity of four polycyclic aromatic hydrocarbons, singly and in combination, to the marine diatom Phaeodactylum tricornutum

Polycyclic aromatic hydrocarbons (PAHs) enter the aquatic environment by various routes and are usually found as mixtures in the water. Many studies have shown that solar ultraviolet (UV) radiation can greatly enhance the toxicity of some PAHs to a variety of marine species. In the present study, we tested the phototoxicity of four PAHs with simple structures, both alone and in binary combinations, to a species of marine diatom, Phaeodactylum tricornutum, in the laboratory. The results indicated that simulated solar UV radiation not only enhanced the toxicity of the different PAHs to this alga, but also changed their relative toxic strengths. The photo-induced toxicity of PAHs to this alga might be a synergistic effect of photo-modification and photosensitization reactions, causing the microalgal cells to suffer oxidative stress. Four binary mixtures of these PAHs were found to have a synergistic joint action mode, while two binary mixtures displayed an antagonistic reaction, revealing a complex pattern of possible interactions of PAHs with marine diatoms.     

Predicting survival of grass shrimp (Palaemonetes pugio) exposed to naphthalene, fluorene, and dibenzothiophene

The composition and persistence of dissolved polycyclic aromatic hydrocarbons (PAHs) released to the water column during oil spills are altered by weathering, tidal transport, and addition of dispersants. Conventional toxicity effect metrics, such as the median lethal concentration (LC50), are inaccurate predictors of mortality from all toxicant exposure duration/concentration combinations likely to occur during spills. In contrast, survival models can predict the proportions of animals dying as a consequence of exposures differing in duration and intensity. Extending previous work with ethylnaphthalene, dimethylnaphthalene, and phenanthrene, survival time models were developed that include exposure duration and concentration to predict time to death for grass shrimp (Palaemonetes pugio). Two additional PAHs (naphthalene and fluorene) and a heterocyclic aromatic hydrocarbon (dibenzothiophene) were evaluated for the present study. Preliminary explorations of these models confirmed that quantitative structure- activity regression models were possible for predicting survival model parameters from compound characteristics. Conventional 48-h LC50s also were calculated for the compounds and combined with published LC50s to predict relative PAH toxicity to P. pugio based on octanol-water partitioning.     

Ecotoxicity of Sediments Contaminated by the Oil Spill Associated with the Tanker “Prestige” Using Juveniles of the Fish Sparus aurata

In November 2002, the oil spill from the tanker Prestige in the Galician Coast caused an ecological catastrophe in Spain. The adverse effects associated with the contaminants bound to sediments were tested using juveniles of the fish Sparus aurata (seabream). The approach evaluates sediment quality by using an integrated assessment including chemical and ecotoxicological data. Sediment samples were physicochemically characterized, and the concentration of contaminants (polycyclic aromatic hydrocarbons—(PAHs) and metals) was measured. Different biomarkers of exposure (metallothioneins and ethoxyresorufin O-deethylase activity (EROD)) and biomarkers of effect (histopathology) were analyzed along the time. A multivariate analysis approach was used to correlate concentration of contaminants and sublethal effects measured in individuals of fish. Results show that increasing concentrations of PAHs in sediments were related to increased EROD activities and histopathological lesions. This is the first evidence showing adverse effects associated with petroleum contamination of PAHs in sediments after this spill, and it demonstrates the utility of the sublethal toxicity tests for monitoring the impact of petroleum spills.     

Quantifying the concentration of crude oil microdroplets in oil-water preparations

Dissolved constituents of crude oil, particularly polycyclic aromatic hydrocarbons (PAHs), can contribute substantially to the toxicity of aquatic organisms. Measured aqueous concentrations of high-molecular weight PAHs (e.g., chrysenes, benzo[a]pyrene) as well as long-chain aliphatic hydrocarbons can exceed the theoretical solubility of these sparingly soluble compounds. This is attributed to the presence of a "microdroplet" or colloidal oil phase. It is important to be able to quantify the dissolved fraction of these compounds in oil-in-water preparations that are commonly used in toxicity assays because the interpretation of test results often assumes that the compounds are dissolved. A method is presented to determine the microdroplet contribution in crude oil-in-water preparations using a comparison of predicted and measured aqueous concentrations. Measured concentrations are reproduced in the model by including both microdroplets and dissolved constituents of petroleum hydrocarbons. Microdroplets were found in all oil-water preparation data sets analyzed. Estimated microdroplet oil concentrations typically ranged from 10 to 700 μg oil/L water. The fraction of dissolved individual petroleum hydrocarbons ranges from 1.0 for highly soluble compounds (e.g., benzene, toluene, ethylbenzene, and xylene) to far less than 0.1 for sparingly soluble compounds (e.g., chrysenes) depending on the microdroplet oil concentration. The presence of these microdroplets complicates the interpretation of toxicity test data because they may exert an additional toxic effect due to a change in the exposure profile. The implications of the droplet model on toxicity are also discussed in terms of both dissolved hydrocarbons and microdroplets.     

Can Crude Oil Toxicity on Phytoplankton Be Predicted Based on Toxicity Data on Benzo(a)Pyrene and Naphthalene?

Polycyclic aromatic hydrocarbons (PAHs), which are major components of crude oil, are responsible in large part for the toxicity of crude oil to phytoplankton. This study addressed the following question. Can reliable predictions of the aquatic toxicity of crude oil, a multi-component mixture, be described from toxicity data on individual PAH compounds? Naphthalene, the most abundant PAH compound, and benzo(a)pyrene, a highly toxic PAH compound, were selected as model compounds to quantify toxicity of crude oil on two phytoplankton species, Ditylum brightwellii and Heterocapsa triquetra, by analyzing the effects of different concentrations of these PAHs on growth rate. EC₅₀ values suggested that the diatom D. brightwellii was more vulnerable to both toxicants than the dinoflagellate H. triquetra. However, a previous study, which investigated the impact of crude oil on the same two species, had opposite results. The differences in response from these phytoplankton species to naphthalene and benzo(a)pyrene toxicity compared to their response to crude oil suggest that they may not be solely used as surrogates to assess crude oil toxicity on phytoplankton.