Cytotoxicity and enzymatic biomarkers as early indicators of benthic responses to the soluble-fraction of diesel oil.

Xenobiotics from oil tanker leaks and industrial discharges are amongst the main human impacts to confined coastal areas. We assessed the genotoxic responses to the water-soluble fraction of diesel oil in the polychaete Laeonereis culveri and the bivalve Anomalocardia flexuosa, two widespread benthic species in subtropical estuaries from the Southwestern Atlantic. We hypothesized that the highest responsiveness would be expressed by significantly different biomarkers responses between control and oil-impacted treatments. Responsiveness to diesel oil was investigated using an experimental design with two fixed factors (contaminant percentages and times of exposure). After exposure, we monitored the responses of the oxidative stress enzymes and performed micronuclei tests. Results were congruent for both species. Antioxidant defense of glutathione S-transferase and the induction of micronuclei and nuclear buds, the latter just for the bivalve, were significantly affected by polycyclic aromatic hydrocarbons, with significant increases on the seventh day of exposure and in the higher concentrations, compared to controls groups. We assessed the benefits and drawbacks of using each biomarker in laboratory experiments. Both species are indicators of early, and rapid responses to genotoxic contaminants in subtropical estuarine habitats. We suggest that the micronuclei frequency in A. flexuosa is a simple, fast and cheap test for genotoxicity in oil-impacted areas. Such early biomarkers are needed to develop better protocols for impact assessment and monitoring under real field conditions.

Biomarker responses in fish exposed to polycyclic aromatic hydrocarbons (PAHs): Systematic review and meta-analysis.

Biomarkers of antioxidant and biotransformation systems are commonly used to assess polycyclic aromatic hydrocarbons (PAHs) pollution in fish. Despite their extensive application of biomarkers, contradictory results are vastly reported in the literature, even for the same species in similar contamination scenarios. This study aims to verify response patterns of biomarkers in fish exposed to PAHs. Through systematic reviews and meta-analyses, we were able to evaluate: (i) overall magnitude of PAHs effects on biotransformation and oxidative stress biomarkers; (ii) patterns of response among experimental approaches (laboratory, field and active biomonitoring), environment (marine and freshwater) and fish habitat (pelagic, demersal, etc.); (iii) effects of exposure route, time and concentration of PAHs; and (iv) which biomarkers respond best to PAHs exposure. Overall, biomarker responses were significantly affected by PAHs exposure. The activities of ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), superoxide dismutase (SOD), glutathione peroxidase (GPx) and levels of oxidized glutathione (GSSG) and lipid peroxide (LPO) significantly increased in fish exposed to PAHs, whereas catalase (CAT) and glutathione reductase (GR) activities and levels of reduced glutathione (GSH) were not affected. Amongst responsive biomarkers, EROD and GST activities significantly differed among approaches and between marine and freshwater environments, but were not affected by fish habitat. GSSG levels were higher in fish from laboratory bioassays compared to the field, but did not differ between environments nor habitats. Exposure route played a major role only for GST and GPx responses. Finally, increasing PAHs concentration and exposure time had a significant effect on all assessed biomarkers, except for CAT. We conclude that EROD and GST are robust biomarkers to assess PAHs effects in fish. Contrarily, CAT is an inadequate biomarker of PAHs exposure since no significant response was observed. Our study also highlighted some research gaps in PAHs contamination studies, such as a clear lack of active biomonitoring experiments.

Growth, Toxin Production and Allelopathic Effects of Pseudo-nitzschia multiseries under Iron-Enriched Conditions.

In order to assess the effects of Fe-enrichment on the growth and domoic acid (DA) production of the toxigenic diatom Pseudo-nitzschia multiseries, static cultures that received the addition of different iron (Fe) concentrations were maintained for 30 days. Intra- and extracellular DA concentrations were evaluated over time, and growth and chain-formation were compared to those of non-toxic diatoms, Bacillaria sp. Growth rates of P. multiseries (µ = 0.45-0.73 d-1) were similar among cultures containing different Fe concentrations. Likewise, the similar incidence and length of P. multiseries stepped cell chains (usually 2-4; up to 8-cell long) among the treatments reinforces that the cultures were not growth-inhibited under any condition tested, suggesting an efficient Fe acquisition mechanism. Moreover, DA concentrations were significantly higher under the highest Fe concentration, indicating that Fe is required for toxin synthesis. Bacillaria sp. reached comparable growth rates under the same Fe concentrations, except when the dissolved cell contents from a P. multiseries culture was added. The 50-70% reduction in cell density and 70-90% decrease in total chlorophyll-a content of Bacillaria sp. at early stationary growth phase indicates, for the first time, an allelopathic effect of undetermined compounds released by Pseudo-nitzschia to another diatom species.

How and where to perform biomonitoring studies: different levels of toxic metal pollution are detected in the Alagados Reservoir in Southern Brazil.

The Alagados Reservoir located in the Brazilian city of Ponta Grossa is used to supply water for human consumption but is impacted by toxic metals. The current study combined chemical, biochemical, and multivariate analyses to determine the bioavailability of toxic metals at three sites (AL-A, AL-B, and AL-C) within the Alagados Reservoir. Metal bioaccumulation was analyzed in the liver, gills, and muscle tissue of a native fish species (Geophagus brasiliensis), and neurotoxicity, xenobiotic metabolism, and oxidative stress were evaluated using biochemical biomarkers. Additionally, histopathological studies were performed on the gills and the liver using scanning electron microscopy (SEM) and conventional light microscopy (LM), respectively. Overall, the bioaccumulation of metals, biomarkers of oxidative stress, and melanomacrophage counts indicate that the AL-C and the AL-A sites are the most and least affected by metals, respectively. The AL-B site presented the lowest acetylcholinesterase enzyme activity, a finding which was probably associated with the agricultural activities around this area of the reservoir. The biomarkers clearly revealed that toxic metals negatively affect all three sites studied herein and that human activity is the major source of pollutants. Despite the existence of different pollution levels within the Alagados Reservoir, it is still used as a human water supply.

Oxidative stress in two tropical species after exposure to diesel oil.

Recent offshore petroleum exploration has increased the risks of oil spills worldwide. We investigated biomarker responses to diesel oil exposure in two tropical and subtropical species, the clam Anomalocardia flexuosa and the polychaete Laeonereis culveri. Animals were exposed to oil-spiked sediment at two different concentrations (0.5 L and 1.0 L m-2). Activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx); glutathione transferase (GST); and lipid peroxides (LPO) were assessed in bivalve digestive glands and polychaete whole-body homogenates at 36 and 60 h of exposure. Significant variation in enzymatic antioxidant activity depended on the sampling time after exposure. No similar response patterns, either increases or decreases, were detected for the two target species, and biomarker responses were species-specific. L. culveri showed clearer patterns in its antioxidant response and should be prioritized over other species in biomonitoring studies involving oil exposure. Understanding the temporal variability of these biomarkers is a necessary action before implementing them as indicators measures in oil contamination biomonitoring programs. Our results provide a better understanding of biomarker responses in subtropical species, evidencing their potential use as sentinels of oil contamination.

Antioxidant responses in estuarine invertebrates exposed to repeated oil spills: Effects of frequency and dosage in a field manipulative experiment.

We have experimentally investigated the effects of repeated diesel spills on the bivalve Anomalocardia brasiliana, the gastropod Neritina virginea and the polychaete Laeonereis culveri, by monitoring the responses of oxidative stress biomarkers in a subtropical estuary. Three frequencies of exposure events were compared against two dosages of oil in a factorial experiment with asymmetrical controls. Hypotheses were tested to distinguish between (i) the overall effect of oil spills, (ii) the effect of diesel dosage via different exposure regimes, and (iii) the effect of time since last spill. Antioxidant defense responses and oxidative damage in the bivalve A. brasiliana and the polychaete L. culveri were overall significantly affected by frequent oil spills compared to undisturbed controls. The main effects of diesel spills on both species were the induction of SOD and GST activities, a significant increase in LPO levels and a decrease in GSH concentration. N. virginea was particularly tolerant to oil exposure, with the exception of a significant GSH depletion. Overall, enzymatic activities and oxidative damage in A. brasiliana and L. culveri were induced by frequent low-dosage spills compared to infrequent high-dosage spills, although the opposite pattern was observed for N. virginea antioxidant responses. Antioxidant responses in A. brasiliana and L. culveri were not affected by timing of exposure events. However, our results revealed that N. virginea might have a delayed response to acute high-dosage exposure. Experimental in situ simulations of oil exposure events with varying frequencies and intensities provide a useful tool for detecting and quantifying environmental impacts. In general, antioxidant biomarkers were induced by frequent low-dosage exposures compared to infrequent high-dosage ones. The bivalve A. brasiliana and the polychaete L. culveri are more suitable sentinels due to their greater responsiveness to oil and also to their wider geographical distribution.

Are intertidal soft sediment assemblages affected by repeated oil spill events? A field-based experimental approach.

This study investigates the impact of repeated diesel spills on the structure of intertidal macrofaunal assemblages of a subtropical estuary. Three frequencies of exposure events were compared against two dosages of oil in a factorial experiment with asymmetrical controls. Hypotheses were tested to distinguish between (i) the overall effect of oil spills, (ii) the effect of diesel dosage via different exposure regimes, and (iii) the effect of time since last spill. Repeated oil spills dramatically altered the overall structure of assemblages and reduced the total density of macrofauna and densities of dominant taxa. Increasing the frequency of oil spills negatively affected macrofauna. In general, frequent low-dosage oil spills were more deleterious than infrequent high-dosage ones. However, increases in densities of some taxa, mainly the gastropod Heleobia australis, were observed in response to infrequent spills. Our results highlight the importance of repeated exposure events in determining the extent of oil impacts.

Antioxidant defense responses in Mytella guyanensis (Lamarck, 1819) exposed to an experimental diesel oil spill in Paranaguá Bay (Paraná, Brazil).

We evaluated the effects of diesel oil on the bivalve Mytella guyanensis using biomarkers of oxidative stress (glutathione S-transferase, glutathione peroxidase, and reduced glutathione) after an experimental in situ spill in a mangrove area in southern Brazil. A linear model was developed for the Multiple Before-After Control-Impact (MBACI) experimental design to assess the significance of biological responses. Control and impacted sites were sampled seven and two days before as well as two and seven days after the spill. With the exception of a late response of reduced glutathione (GSH) levels on day seven, none of the biomarkers were significantly altered by the impact. This result was attributed to the high environmental variability of the experimental sites combined with a low sensitivity of Mytella guyanensis to diesel oil at short time-scales. The high resistance of M. guyanensis suggests that its antioxidant response is triggered only after a medium- to long-term exposure to contaminants.

Is the cutting of oil contaminated marshes an efficient clean-up technique in a subtropical estuary?

Cutting and removal of oil-impacted marsh plants are still used worldwide as a clean-up and recovery technique. To experimentally test the efficacy of cutting and removing marsh plants under subtropical conditions, we simulated an oil spill (Bunker MF-180) in Spartina alterniflora marshes and compared the responses of plant height, biomass, density of culms and number of flowering plants in high and low energy areas in Paranaguá Bay (S Brazil) for about 9 months. Cutting and removal were inefficient in promoting or accelerating the recovery of the impacted areas. Cut or uncut impacted marshes fully recovered within 6 months, both in low and high energy areas. Plant cutting should be practiced only when there is an effective risk of contamination of groundwater near urban areas, when obvious aesthetical issues are involved in areas of touristic interest or when there are real short-term conservation risks to threatened species.