Maria João Botelho

Emissions of metals and polychlorinated dibenzo(p)dioxins and furans (PCDD/Fs) from Portland cement manufacturing plants: Inter-kiln variability and dependence on fuel-types

Emissions from Portland cement manufacturing facilities may increase health risks in nearby populations and are thus subject to stringent regulations. Direct testing of pollutant concentrations in exhaust gases provides the best basis for assessing the extent of these risks. However, these tests (i) are often conducted under stressed, rather than typical, operating conditions, (ii) may be limited in number and duration, and (iii) may be influenced by specific fuel-types and attributes of individual kilns. We report here on the results of more than 150 emissions-tests conducted of two kilns at a Portland cement manufacturing plant in Portugal. The tests measured various regulated metals and polychlorinated dibenzo(p)dioxins and furans (PCDD/Fs). Stack-gas concentrations of pollutants were found to be highly variable, with standard deviations on the order of mean values. Emission rates of many pollutants were higher when coal was used as the main kiln fuel (instead of petroleum coke). Use of various supplemental fuels, however, had little effect on stack emissions, and few statistically significant differences were observed when hazardous waste was included in the fuel mix. Significant differences in emissions for some pollutants were observed between the two kilns despite their similar designs and uses of similar fuels. All measured values were found to be within applicable regulatory limits.

Depuration kinetics of paralytic shellfish toxins in Mytilus galloprovincialis exposed to Gymnodinium catenatum: laboratory and field experiments.

The kinetics of paralytic shellfish toxins in Mytilus galloprovincialis, previously exposed to Gymnodinium catenatum, was studied under depuration laboratory conditions and over a declining bloom of the dinoflagellate in the field. The variation of the levels observed throughout the laboratory experiment was characterized by a fast depuration of B1, C1 + 2, dcSTX and dcGTX2 + 3, possibly due to the gut evacuation of unassimilated toxins or microalgae cells, or loss during digestive mechanisms. Subsequent enhancements were observed for all compounds with emphasis to dcSTX and dcGTX2 + 3, pointing to biotransformation of the assimilated toxins. Then levels decreased gradually. A first-order depuration kinetic model fitted well to the decrease of B1, C1 + 2 and dcGTX2 + 3 concentrations, but not for dcSTX. Mussels exposed to a declining bloom of Gymnodinium catenatum exhibited a loss of toxins following the same pattern. Despite the low abundance of this dinoflagellate, a similar kinetic model was applied to the field data. The depuration rate of dcGTX2 + 3 in the field experiment (0.153 ± 0.03 day(-1)) significantly exceeded the value calculated in the laboratory (0.053 ± 0.01 day(-1)), while smaller differences were obtained for B1 (0.071 ± 0.02 and 0.048 ± 0.01 day(-1)) and similar values for C1 + 2 (0.082 ± 0.03 and 0.080 ± 0.03 day(-1)). The slower depuration rate of dcGTX2 + 3 in the heavily contaminated mussels at the laboratory may be related to a more effective contribution of C1 + 2 biotransformation.

Matrix effect on paralytic shellfish toxins quantification and toxicity estimation in mussels exposed to Gymnodinium catenatum

Paralytic shellfish toxins were quantified in whole tissues of the mussel Mytilus galloprovincialis exposed to blooms of the dinoflagellate Gymnodinium catenatum in Portuguese coastal waters. A validated liquid chromatography method with fluorescence detection, involving pre-chromatographic oxidation was used to quantify carbamoyl, N-sulfocarbamoyl and decarbamoyl toxins. In order to test for any matrix effect in the quantification of those toxins, concentrations obtained from solvent and matrix matched calibration curves were compared. A suppression of the fluorescence signal was observed in mussel extract or fraction in comparison to solvent for the compounds dcGTX2 + 3, GTX2 + 3 and GTX1 + 4, while an enhancement was found for C1 + 2, dcSTX, STX, B1, dcNEO and NEO. These results showed that a matrix effect varies among compounds. The difference of concentrations between solvent and matrix matched calibration curves for C1 + 2 (median = 421 ng g−1) exceeded largely the values for the other quantified compounds (0.09–58 ng g−1). Those differences were converted into toxicity differences, using Oshima toxicity equivalence factors. The compounds C1 + 2 and dcNEO were the major contributors to the differences of total toxicity in the mussel samples. The differences of total toxicity were calculated in ten mussel samples collected during a 10-week blooming period in Portuguese coastal lagoon. Values varied between 53 and 218 µg STX equivalents kg−1. The positive differences mean that the estimated toxicity using solvent calibration curves exceed the values taking into account the matrix. For the toxicity interval 200–800 µg STX equivalents kg−1 an increase was found between 44 and 28%.

Uptake and release of paralytic shellfish toxins by the clam Ruditapes decussatus exposed to Gymnodinium catenatum and subsequent depuration

A laboratory experiment was performed with the clam Ruditapes decussatus, fed with the toxic dinoflagellate Gymnodinium catenatum and the non-toxic algae Isochrysis galbana (14 days) and subsequently only with I. galbana (15 days). Individual paralytic shellfish toxins were determined by LC-FLD in G. catenatum cells, whole clam tissues, and particulate organic matter (POM) produced by clams. The toxins dcSTX and dcGTX2 + 3 in the algae were less abundant than C1 + 2 and B1, but were predominant in clams during both the exposure and depuration phases. The toxin dcNEO was only detected in clams during a short period, indicating conversion from other compounds. The toxin composition of the POM indicated the export of dcSTX as faeces or pseudo-faeces along the entire experiment (2.5-14 nmol mg(-1)), B1 was present in a short period of the exposure and C1 + 2 and dcGTX2 + 3 absent. A mass balance calculation indicated that approximately 95% of C1 + 2 and 85% of B1 supplied to the clams were converted into other toxins or lost in solution. Conversely, the net gain of 512, 61 and 31 nmol for dcSTX, dcGTX2 + 3 and dcNEO, respectively, suggests the conversion from other assimilated compounds by clams during exposure and depuration phases.

Tracking the Spatial Fate of PCDD/F Emissions from a Cement Plant by Using Lichens as Environmental Biomonitors.

In an area with multiple sources of air pollution, it is difficult to evaluate the spatial impact of a minor source. Here, we describe the use of lichens to track minor sources of air pollution. The method was tested by transplanting lichens from a background area to the vicinity of a cement manufacturing plant that uses alternative fuel and is located in a Natural Park in an area surrounded by other important sources of pollution. After 7 months of exposure, the lichens were collected and analyzed for 17 PCDD/F congeners. The PCDD/F profiles of the exposed lichens were dominated by TCDF (50%) and OCDD (38%), which matched the profile of the emissions from the cement plant. The similarity in the profiles was greatest for lichens located northeast of the plant (i.e., in the direction of the prevailing winds during the study period), allowing us to evaluate the spatial impact of this source. The best match was found for sites located on the tops of mountains whose slopes faced the cement plant. Some of the sites with highest influence of the cement plant were the ones with the highest concentrations, whereas others were not. Thus, our newly developed lichen-based method provides a tool for tracking the spatial fate of industrially emitted PCDD/Fs regardless of their concentrations. The results showed that the method can be used to validate deposition models for PCDD/F industrial emissions in sites with several sources and characterized by complex orography.

Changes of paralytic shellfish toxins in gills and digestive glands of the cockle Cerastoderma edule under post-bloom natural conditions.

Concentrations of the paralytic shellfish toxins C1+2, C3+4, GTX5, GTX6, dcGTX2+3, dcSTX, dcNEO, GTX2+3, GTX1+4, STX and NEO were determined by LC-FLD in composite samples of digestive glands and gills of Cerastoderma edule cockle. The specimens were sampled in Aveiro lagoon, Portugal, under natural depuration conditions (days 0, 8, 12, 14, 19, 21 and 25) after exposure to a bloom of Gymnodinium catenatum. Individual paralytic shellfish toxins indicated different pathways of elimination and biotransformation in digestive gland and gills. Toxin concentrations in gills were lower than in digestive gland. Most of the quantified toxins in digestive gland decreased during the 25 days of observation according to negative exponential curves, and only GTX5, GTX6 and NEO showed slight irregularities with time. Concentrations of C1+2, C3+4 and dcGTX2+3 in gills decreased progressively, however GTX5, GTX6 and dcSTX showed pronounced increases. Higher concentrations of those toxins in days 8 and 12 in comparison to the initial value (day 0) indicate conversion of other toxins into GTX5, GTX6 and dcSTX during those periods. It appears that inter-conversion of toxins occurs as G. catenatum cells are retained in gills before being transferred to other compartments.

Profiles of paralytic shellfish toxins in bivalves of low and elevated toxicities following exposure to Gymnodinium catenatum blooms in Portuguese estuarine and coastal waters.

Profiles of paralytic shellfish toxins (PSTs) were examined in 405 composite samples of Mytilus spp., Cerastoderma edule, Donax trunculus and Spisula solida collected between 2007 and 2012 from natural production areas in two estuaries (Aveiro and Mondego), two coastal lagoons (Óbidos and Formosa), and three open coastal areas (Aguda, Comporta and Culatra). Toxin concentrations were obtained from the biotoxin monitoring programme database. Episodes of PST toxicity in Portugal have been associated with Gymnodinium catenatum blooms. Toxin profiles for each species showed no trend over the surveyed years. In general, profiles differ only slightly among areas, except for Óbidos. However, toxin profiles in bivalves varied between low and elevated toxicities, corresponding to below and above the PST regulatory limit, respectively. The ratio R1=(C1+2):B1, which were the main toxins produced by G. catenatum cells, decreased considerably between elevated and low toxicity cockles, indicating the elimination of C1+2 or conversion of compounds into B1. R2=[(dcSTX)+(dcGTX2+3)]:[(C1+2)+(B1)], which represents the ratio of minor to major toxins in G. catenatum cells, increased substantially in wedge clams (D. trunculus) of low toxicity and less markedly in cockles (C. edule) and mussels (Mytilus spp.). These differences are interpreted as the predominance of a biotransformation phase after exposure to the algal bloom. The toxin profile of surf clams (S. solida) was dominated by decarbamoyl compounds, reflecting intense biotransformation during exposure to blooms. The higher ratio R2 in low toxicity samples suggests that elimination of the produced decarbamoyl toxins was slower than biotransformation.

Determination of paralytic shellfish toxins using potentiometric electronic tongue

The present work deals with the application of an electronic tongue based on potentiometric chemical sensors to the detection of paralytic shellfish toxins (PSTs) in bivalve extracts. PSTs are naturally occurring toxic compounds produced by some phytoplankton species that may accumulate in bivalves during algal blooms. Bivalves for human consumption are therefore monitored for PSTs. Electronic tongue comprised six miniaturized sensors with solid inner contact and plasticized polyvinylchloride membranes. Calibration models were calculated by PLS regression using measurements in sixteen model mixed solutions containing four PSTs commonly detected in bivalves harvested in the Portuguese coast. Transfer of the calibration models to sample matrix was done by joint-PLS regression using measurements in five mussel extracts spiked with PST standards. Quantification of PSTs in extracts of naturally contaminated mussel using electronic tongue and updated calibration model was in agreement with measurements by the reference chromatographic method.

Partitioning of paralytic shellfish toxins in sub-cellular fractions of the digestive gland of the cockle Cerastoderma edule: Changes under post-bloom natural conditions.

Concentrations of paralytic shellfish toxins (C1+2, B1, dcGTX2+3, dcSTX, GTX2+3 and STX) were determined by LC-FLD in composite samples of digestive glands of the cockle Cerastoderma edule and in each sub-cellular particulate fractions obtained after differential centrifugation (nuclei+debris, mitochondria, lysosomes and microsomes). The specimens were sampled during the exposure to a bloom of Gymnodinium catenatum (day 0) and in the subsequent 8, 12, 14, 19, 21 and 25 days under natural depuration conditions. Toxin profiles of digestive glands were dominated by C1+2 followed by B1 and dcGTX2+3, although the proportion between C1+2 and B1 contents decreased with the time, indicating a slower elimination of B1. All toxins, except GTX2+3 and STX, were quantified in the four sub-cellular fractions. The content of the quantified toxins decreased most markedly in nuclei+debris and microsomal fractions, during the first eight and 12 days, respectively. Conversely, different patterns were observed among toxins in mitochondrial and lysosomal fractions. The less accentuated decreases of dcGTX2+3 and dcSTX contents in the mitochondrial fraction may have resulted from the conversion of other toxins, like C1+2 and B1, associated with enzymatic activities in that fraction. The largest discrepancy was registered in lysosomal fraction for B1, since its content increased after eight days of post-bloom conditions. Input of B1 may come from the conversion of other toxins, like the abundant B2 and C1+2. These transformations are associated to the major role of lysosomes in the intra-cellular digestive process of materials acquired through vesicular transport.

Nutrients and clam contamination by Escherichia coli in a meso-tidal coastal lagoon: Seasonal variation in counter cycle to external sources

The clam Ruditapes decussatus was transplanted from a natural recruitment area of Ria Formosa to three sites, surveyed for nutrients in water and sediments. Specimens were sampled monthly for determination of Escherichia coli, condition index and gonadal index. Higher nutrient values in low tide reflect drainage, anthropogenic sources or sediment regeneration, emphasising the importance of water mixing in the entire lagoon driven by the tide. Despite the increase of effluent discharges in summer due to tourism, nutrient concentrations and E. coli in clams were lower in warmer periods. The bactericide effect of temperature and solar radiation was better defined in clams from the inlet channel site than from sites closer to urban effluents. High temperature in summer and torrential freshwater inputs to Ria Formosa may anticipate climate change scenarios for south Europe. Seasonal variation of nutrients and clam contamination may thus point to possible alterations in coastal lagoons and their ecosystem services.