Biochemical effects in crabs (Carcinus maenas) and contamination levels in the Bizerta Lagoon: an integrated approach in biomonitoring of marine complex pollution

The biochemical effects in Carcinus maenas and contamination levels in seawater and sediments of Bizerta Lagoon (northeast of Tunisia) were investigated. The levels of metals and hydrocarbons were higher in seawater and sediments in Menzel Bourguiba and Cimentery in February and July than in the other sampling sites. Differences among sites for glutathione S-transferase, catalase, acetylcholinesterase activities, and the content of lipid peroxidation and metallothioneins in two important organs which accumulated contaminants (the gills and the digestive gland) of the C. maenas were found and possibly related to differences in metal and hydrocarbon levels. The seasonal variation of biomarkers was possibly associated with chemical contamination and also with the high fluctuation of physico-chemical characteristics of the sites. The integrated biomarker response values found in the five sites is in good agreement with hydrocarbon and trace metal concentrations detected in the water and sediments of the stressful places where crabs are living.     

Diversity and community structure of culturable arsenic-resistant bacteria across a soil arsenic gradient at an abandoned tungsten-tin mining area

We studied the bacterial diversity at a single location (the Terrubias mine; Salamanca province, Spain) with a gradient of soil As contamination to test if increasing levels of As would (1) change the preponderant groups of arsenic-resistant bacteria and (2) increase the tolerance thresholds to arsenite [As(III)] and arsenate [As(V)] of such bacteria. We studied the genetic and taxonomic diversity of culturable arsenic-resistant bacteria by PCR fingerprinting techniques and 16S rRNA gene sequencing. Then, the tolerance thresholds to As(III) and As(V) were determined for representative strains and mathematically analyzed to determine relationships between tolerances to As(III) and As(V), as well as these tolerances with the soil contamination level. The diversity of the bacterial community was, as expected, inversely related to the soil As content. The overall preponderant arsenic-resistant bacteria were Firmicutes (mainly Bacillus spp.) followed by γ-Proteobacteria (mainly Pseudomonas spp.), with increasing relative frequencies of the former as the soil arsenic concentration increased. Moreover, a strain of the species Rahnella aquatilis (γ-Proteobacteria class) exhibited strong endurance to arsenic, being described for the first time in literature such a phenotype within this bacterial species. Tolerances of the isolates to As(III) and As(V) were correlated but not with their origin (soil contamination level). Most of the strains (64%) showed relatively low tolerances to As(III) and As(V), but the second most numerous group of isolates (19%) showed increased tolerance to As(III) rather than to As(V), even though the As(V) anion is the prevalent arsenic species in soil solution at this location. To our knowledge, this is the first study to report a shift towards preponderance of Gram-positive bacteria (Firmicutes) related to high concentrations of soil arsenic. It was also shown that, under aerobic conditions, strains with relatively enhanced tolerance to As(III) predominated over the most As(V)-tolerant ones.     

An integrated approach using bioaccumulation and biomarker measurements in female shore crab, Carcinus maenas

The shore crab Carcinus maenas, was used as a biomonitoring organism to measure the potential impact that the mining spill in the Guadalquivir Estuary (SW, Spain), in 1998 may have exerted on local biota. Female intermoult C. maenas were exposed to dissolved cadmium, copper and zinc at concentrations found in local waters following the spill (3 microgl-1 Cd, 15 microgl-1 Cu, 700 microgl-1 Zn). Groups of shore crabs were exposed for 21 days to these metals, both singularly and in combination. Residues of metals were measured in gill, midgut gland and muscle tissues, together with metallothioneins in the midgut gland, at the end of the exposure period. Haemolymph samples were taken every seven days, and vitellogenin/vitellin analysed to determine the flux of these compounds. Biomarker results suggested that they would be good indicators of the effects of heavy metal environmental contamination, particularly in the case of Cd and Zn. Variation in results was obtained between responses measured in crabs exposed to single metals and those exposed to combinations of metals.     

Application of biomarkers to assess the condition of European Marine Sites

A series of European Marine Sites has been designated as Special Areas of Conservation (SAC) in England. The aim of this study was to develop a practical methodology to assess the condition of SACs by applying a suite of biomarkers. Biomarkers were applied to the blue mussel Mytilus edulis and the shore crab Carcinus maenas from the Fal and Helford SAC (Cornwall). Individual biomarkers provided useful diagnostic information on the activity of certain classes of contaminants and an integrated Biomarker Response Index (BRI) was used to achieve a more holistic understanding of the condition of the SAC. The BRI indicated that the general health of both organisms was impacted in the upper part of the SAC (Fal Estuary) which correlated well with known chemical hotspots and sources of contamination. The BRI allows a pragmatic way to prioritise SAC sites that may require further investigative studies.     

Metals bioaccumulation and histopathological biomarkers in Carcinus maenas crab from Bizerta lagoon,Tunisia

Metals concentrations and histolopathological lesions of gills and digestive gland were investigated in Carcinus maenas crabs sampled from Bizerta Lagoon and Kuriat Island (Tunisia) as control site. The concentrations of trace metals varied between tissues, sites and sampling time. The highest levels of the analysed metals in gills and digestive gland were noted in Menzel Bourguiba and Cimentery sites at both sampling times (February and July). The higher metals loads were associated with severe and various tissues alterations in contaminated crabs. We particularly noted in the gills a haemocytic infiltration, distension and enlargement of the lamellae, lifting of lamellar epithelium, necrotic lesions and fusion of lamellae in the most polluted sites (Menzel Bourguiba and Cimentery). Moreover, others pathological alterations were observed in digestive gland of crabs collected from polluted sites and with a severity site dependent. We observed necrotic tubules containing tissue debris in the lumen with more intensity in crabs collected from Cimentery site in both sampling times. The thickened basal laminae and the walling off of the tubules by haemocytes around the thickened basal laminae were more abundant at Menzel Bourguiba than at others sites. The coagulation in the thickened basal laminae was observed only at Cimentery in February. Tissues histopathological lesions were sensitive to discriminate crabs of different sites and demonstrated its usefulness in this biomonitoring study. We recommend the association of histopatholocial lesions to biochemical biomarkers in future biomonitoring studies.     

The East African decapod crab Neosarmatium meinerti (de Man) sweeps mangrove floors clean of leaf litter

Leaf litter removal by the abundant mangrove decapod crab Neosarmatium meinerti was studied in series of field and laboratory experiments in East Africa. In the high intertidal Avicennia marina zone crabs buried all leaves placed on the forest floor and consumed on average 67% of them within 2 hrs. High shore crabs in Kenya buried 4 g m(-2) leaf-litter in 1 hr, i.e. approx. twice the daily litter fall. In contrast, in the low shore Sonneratia alba zone, where typical leaf-eating crabs were absent, none of the offered leaves showed signs of herbivory. Leaf choice experiments in the laboratory showed that N. meinerti preferred some species to others. Leaf consumption per gram crab was higher in females than males. The laboratory studies also indicated that crabs could consume substantially more than the average daily litter fall. Video recordings documented frequent fights to gain or retain fallen leaves, suggesting strong competition for leaf litter. Earlier studies indicating that N. meinerti may sweep mangrove forest floors clean of leaf litter are confirmed. In high shore mangroves of East and South Africa where N. meinerti is common, energy flow appears unique: virtually all litter production is retained.     

Copper tolerance in the macrolichens Cladonia furcata and Cladina arbuscula subsp. mitis is constitutive rather than inducible

In this study we assessed the degree of copper (Cu) tolerance in two common lichen species (Cladonia furcata and Cladina arbuscula subsp. mitis) that grow on both uncontaminated substrata and the surface of waste heaps from abandoned old Cu-mines. Regardless of their locality, populations of these lichens contain identical strains of photobionts (Asterochloris clade A in C. arbuscula subsp. mitis and clade D in C. furcata). Therefore, it was expected that if there were differences in Cu toxicity or tolerance between populations, that the photobiont could not be a key element of Cu tolerance in these two lichen species. In laboratory experiments samples of both lichen species (from contaminated and control sites) were incubated in Cu solutions (500 μM) for 24 h. We attempted to determine whether Cu tolerance in these lichens was constitutive, or inducible form. Based on measurements of Cu accumulation, chlorophyll a integrity, chlorophyll a fluorescence, photosynthesis, respiration, measurements of the content of thiobarbituric acid reactive substances (TBARS), the content of soluble proteins, reactive oxygen species (ROS) and the amount of extracellular secondary metabolites of both lichens we found that there were no significant differences in the response of all selected populations of both lichen species to short-term exposure to these high levels of Cu. As a result, we conclude that Cu tolerance in these two lichen species is the constitutive rather than the inducible.     

Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus

Thirty-nine white-rot fungi belonging to nine species of Agaricomycotina (Basidiomycota) were initially screened for their ability to decrease olive-mill wastewater (OMW) phenolics. Four strains of Ganoderma australe, Ganoderma carnosum, Pleurotus eryngii and Pleurotus ostreatus, were selected and further examined for key-aspects of the OMW biodegradation process. Fungal growth in OMW-containing batch cultures resulted in significant decolorization (by 40-46% and 60-65% for Ganoderma and Pleurotus spp. respectively) and reduction of phenolics (by 64-67% and 74-81% for Ganoderma and Pleurotus spp. respectively). COD decrease was less pronounced (12-29%). Cress-seeds germination increased by 30-40% when OMW was treated by Pleurotus strains. Toxicity expressed as inhibition of Aliivibrio fischeri luminescence was reduced in fungal-treated OMW samples by approximately 5-15 times compared to the control. As regards the pertinent enzyme activities, laccase and Mn-independent peroxidase were detected for Ganoderma spp. during the entire incubation period. In contrast, Pleurotus spp. did not exhibit any enzyme activities at early growth stages; instead, high laccase (five times greater than those of Ganoderma spp.) and Mn peroxidases activities were determined at the end of treatment. OMW decolorization by Ganoderma strains was strongly correlated to the reduction of phenolics, whereas P. eryngii laccase activity was correlated with the effluent’s decolorization.     

Differential Legionella spp. survival between intracellular and extracellular forms in thermal spring environments

Legionella are commonly found in natural and man-made aquatic environments and are able to inhabit various species of protozoa. The relationship between the occurrence of Legionella spp. within protozoa and human legionellosis has been demonstrated; however, the proportions of intracellular and extracellular Legionella spp. in the aquatic environment were rarely reported. In this study, we developed a new method to differentiate intracellular and extracellular Legionella spp. in the aquatic environment. Water samples from three thermal spring recreational areas in southeastern Taiwan were collected and analyzed. For each water sample, concurrent measurements were performed for Legionella spp. and their free-living amoebae hosts. The overall detection rate was 32 % (16/50) for intracellular Legionella spp. and 12 % (6/50) for extracellular Legionella spp. The most prevalent host of Legionella spp. was Hartmannella vermiformis. The identified Legionella spp. differed substantially between intracellular and extracellular forms. The results showed that it may be necessary to differentiate intracellular and extracellular forms of Legionella spp.     

Distribution and trends of mercury in deciduous tree cores

The distribution of total mercury (THg) within common deciduous trees and the applicability of tree cores as biomonitors of historical environmental THg trends were assessed for both contaminated and reference sites around Kingston, Ontario. Samples were collected from Acer spp., Quercus spp. Populus spp. and Salix spp. Bark and wood THg concentrations were found to be highly correlated whereas soil and wood THg concentrations were not. There were no temporal relationships for THg in dated tree rings corresponding with any other known environmental Hg trends. The shoreline speciess, Populus and Salix spp., had the greatest bark and wood Hg concentrations reaching 18 ng/g, significantly higher than for inland trees Quercus and Acer spp. with maximum values of 7 and 1.2 ng/g for bark and wood respectively. While tree cores cannot be reliably used as temporal THg biomonitors, there is promise for tree species such as Populus spp and Salix spp as spatial indicators of local long-term Hg contamination.     

Dechlorination and organohalide-respiring bacteria dynamics in sediment samples of the Yangtze Three Gorges Reservoir

Several groups of bacteria such as Dehalococcoides spp., Dehalobacter spp., Desulfomonile spp., Desulfuromonas spp., or Desulfitobacterium spp. are able to dehalogenate chlorinated pollutants such as chloroethenes, chlorobenzenes, or polychlorinated biphenyls under anaerobic conditions. In order to assess the dechlorination potential in Yangtze sediment samples, the presence and activity of the reductively dechlorinating bacteria were studied in anaerobic batch tests. Eighteen sediment samples were taken in the Three Gorges Reservoir catchment area of the Yangtze River, including the tributaries Jialing River, Daning River, and Xiangxi River. Polymerase chain reaction analysis indicated the presence of dechlorinating bacteria in most samples, with varying dechlorinating microbial community compositions at different sampling locations. Subsequently, anaerobic reductive dechlorination of tetrachloroethene (PCE) was tested after the addition of electron donors. Most cultures dechlorinated PCE completely to ethene via cis-dichloroethene (cis-DCE) or trans-dichloroethene. Dehalogenating activity corresponded to increasing numbers of Dehalobacter spp., Desulfomonile spp., Desulfitobacterium spp., or Dehalococcoides spp. If no bacteria of the genus Dehalococcoides spp. were present in the sediment, reductive dechlorination stopped at cis-DCE. Our results demonstrate the presence of viable dechlorinating bacteria in Yangtze samples, indicating their relevance for pollutant turnover.     

Cadmium tolerance in six poplar species

Selection of poplar species with greater Cd tolerance and exploiting the physiological mechanisms involved in Cd tolerance are crucial for application of these species to phyto-remediation. The aim of this study is to investigate variation in Cd tolerance among the six poplar species and its underlying physiological mechanisms. Cuttings of six Populus species were cultivated for 10 weeks before exposure to either 0 or 200 μM CdSO₄ for 20 days. Gas exchange in mature leaves was determined by a portable photosynthesis system. Cd concentrations in tissues were analyzed by a flame atomic absorbance spectrometry. Subsequently, Cd amount per plant, bio-concentration factor (BCF) and translocation factor (T _f) were calculated. Nonenzymatic compounds and activities of antioxidative enzymes in tissues were analyzed spectrophotometrically. Cd exposure caused decline in photosynthesis in four poplar species including Populus cathayana (zhonghua 1). Among the six species, P. cathayana (zhonghua 1) displayed the highest Cd concentrations in tissues, the largest Cd amount in aerial parts, the highest BCF in aerial parts and T _f under Cd exposure. Under Cd stress, increases in total soluble sugars in roots but decreases in starch in roots, wood, and leaves of P. cathayana (zhonghua 1) were found. Induced O ₂ ^?? and H₂O₂ production in roots and leaves, and increases in free proline, soluble phenolics, and activities of antioxidative enzymes were observed in P. cathayana (zhonghua 1). Based on results of this pot experiment, it is concluded that P. cathayana (zhonghua 1) is superior to other five species for Cd phyto-remediation, and its well-coordinated physiological changes under Cd exposure confer the great Cd tolerance of this species.     

Organ-wise accumulation of fluoride in Prosopis juliflora and its potential for phytoremediation of fluoride contaminated soil

Fluoride (F) contamination is a global environmental problem, as there is no cure of fluorosis available yet. Prosopis juliflora is a leguminous perennial, phreatophyte tree, widely distributed in arid and semi-arid regions of world. It extensively grows in F endemic areas of Rajasthan (India) and has been known as a “green” solution to decontaminate cadmium, chromium and copper contaminated soils. This study aims to check the tolerance potential of P. juliflora to accumulate fluoride. For this work, P. juliflora seedlings were grown for 75 d on soilrite under five different concentrations of F viz., control, 25, 50, 75 and 100 mg NaF kg⁻¹. Organ-wise accumulation of F, bioaccumulation factor (BF), translocation factor (TF), growth ratio (GR) and F tolerance index (TI) were examined. Plant accumulated high amounts of F in roots. The organ-wise distribution showed an accumulation 4.41 mg kg⁻¹dw, 12.97 mg kg⁻¹dw and 16.75 mg kg⁻¹dw F, in stem, leaves and roots respectively. The results indicated significant translocation of F from root into aerial parts. The bioaccumulation and translocation factor values (>1.0) showed high accumulation efficiency and tolerance of P. juliflora to F. It is concluded that P. juliflora is a suitable candidate for phytoremediation purpose and can be explored further for the decontamination of F polluted soils.     

The origin of speciation: trace metal kinetics over natural water/sediment interfaces and the consequences for bioaccumulation

The speciation of heavy metals was measured over a variety of natural and undisturbed water/sediment interfaces. Simultaneously, two benthic species (oligochaete Limnodrilus spp. and the midge Chironomus riparius) were exposed to these sediments. Under occurring redox conditions, free ion activities of trace metals Cd, Cu, Ni, Pb, and Zn were measured with a chelating exchange technique, while geochemical conditions (i.e., redox) remained in tact. Free ion activities were compared with total dissolved concentrations in pore waters and surface waters in order to relate speciation to bioaccumulation. Limnodrilus spp. and C. riparius have accumulation patterns that could be linked to time-dependent exposure concentrations, expressed as chemical speciation, in the surface water and the sediment's pore water. Concentrations of free metal ions in the overlying surface water, rather than in sediment pore water, proved to be the best predictor for uptake. For the first time, measurements are obtained from sediments without disturbing physical-chemical conditions and thus bioavailability, a major restriction of other studies so far.     

Cd accumulation and phytostabilization potential of dominant plants surrounding mining tailings

The objectives of the present study were to compare nine dominant plant species growing in mine tailings and nonmining areas in terms of biomass and Cd concentrations and to search for Cd accumulation and tolerance. Also, more detailed experiments were conducted on Athyrium wardii using a pot experiment to assure its Cd-accumulation ability and tolerance as a potential phytostabilizer of Cd-polluted soils. Nine dominant plant species growing on Pb/Zn mine tailings and their corresponding nonmining ecotypes were investigated for their potential to phytostabilize Cd. The performance of A. wardii exposed to high levels of Cd was investigated under controlled conditions. A field study revealed that the Cd concentrations in the roots of these plants ranged from 0.21 to 251.07?mg?kg^?1, and the highest concentrations were found in A. wardii, which reached a concentration of 69.78, 251.07, and 126.35?mg?kg^?1 during the early growth stage (May), vigorous growth stage (August), and late growth stage (October), respectively. The Cd concentrations of roots among the nine mining ecotypes were positively correlated with available content of Cd in the rhizosphere soils, whereas a negative correlation was observed in the nonmining ecotypes. A pot experiment showed that the mining ecotype of A. wardii had a higher biomass production and Cd retention capacity in roots than that of the nonmining ecotype. Due to the relatively high tolerance to Cd and the capacity of roots to retain this metal, A. wardii may be useful for the phytostabilization of soils contaminated by Cd.     

Spatial distribution of ciguateric fish in the Republic of Kiribati

Ciguatera is food poisoning caused by human consumption of reef fish contaminated with ciguatoxins (CTXs). The expanding international trade of tropical fish species from ciguatera-endemic regions has resulted in increased global incidence of ciguatera, and more than 50 000 people are estimated to suffer from ciguatera each year worldwide. The Republic of Kiribati is located in the Pacific Ocean; two of its islands, Marakei and Tarawa, have been suggested as high-risk areas for ciguatera. The toxicities of coral reef fish collected from these islands, including herbivorous, omnivorous and carnivorous fish (24% [n = 41], 8% [n = 13] and 68% [n = 117], respectively), were analyzed using the mouse neuroblastoma assay (MNA) after CTX extraction. The MNA results indicated that 156 fish specimens, or 91% of the fish samples, were ciguatoxic (CTX levels >0.01 ng g⁻¹). Groupers and moray eels were generally more toxic by an order of magnitude than other fish species. All of the collected individuals of eight species (n = 3-19) were toxic. Toxicity varied within species and among locations by up to 10 000-fold. Cephalapholis argus and Gymnothorax spp. collected from Tarawa Island were significantly less toxic than those from Marakei Island, although all individuals were toxic based on the 0.01 ng g⁻¹ threshold. CTX concentrations in the livers of individuals of two moray eel species (Gymnothorax spp., n = 6) were nine times greater than those in muscle, and toxicity in liver and muscle showed a strong positive correlation with body weight. The present study provides quantitative information on the ciguatoxicity and distribution of toxicity in fish for use in fisheries management and public health.     

Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy

Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella — where two morphologically similar species impossible to separate by light microscopy were distinguished — in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring.     

Isolation of culturable mycobiota from agricultural soils and determination of tolerance to glyphosate of nontoxigenic Aspergillus section Flavi strains

Glyphosate-based herbicides are extensively used in Argentina's agricultural system to control undesirable weeds. This study was conducted to evaluate the culturable mycobiota [colony forming units (CFU) g^?1 and frequency of fungal genera or species] from an agricultural field exposed to pesticides. In addition, we evaluated the tolerance of A. oryzae and nontoxigenic A. flavus strains to high concentrations (100 to 500 mM – 17,000 to 84,500 ppm) of a glyphosate commercial formulation. The analysis of the mycobiota showed that the frequency of the main fungal genera varied according to the analyzed sampling period. Aspergillus spp. or Aspergillus section Flavi strains were isolated from 20 to 100% of the soil samples. Sterilia spp. were also observed throughout the sampling (50 to 100%). Aspergillus section Flavi tolerance assays showed that all of the tested strains were able to develop at the highest glyphosate concentration tested regardless of the water availability conditions. In general, significant reductions in growth rates were observed with increasing concentrations of the herbicide. However, a complete inhibition of fungal growth was not observed with the concentrations assayed. This study contributes to the knowledge of culturable mycobiota from agricultural soils exposed to pesticides and provides evidence on the effective growth ability of A. oryzae and nontoxigenic A. flavus strains exposed to high glyphosate concentrations in vitro.     

PFAS profiles in three North Sea top predators: metabolic differences among species?

Profiles of seven compounds of perfluoro-alkyl substances (PFASs) were compared among three species of top predators from the Danish North Sea: the white-beaked dolphin (Lagenorhynchus albirostris), the harbor porpoise (Phocoena phocoena), and the harbor seal (Phoca vitulina). The seals had higher total burdens (757.8 ng g^?1 ww) than the dolphins (439.9 ng g^?1 ww) and the porpoises (355.8 ng g^?1 ww), probably a reflection of feeding closer to the shore and thus contamination sources. The most striking difference among the species was the relative contribution of perfluorooctanesulfonamide (PFOSA) to the profiles; the seals (0.1 %) had much lower levels than porpoises (8.3 %) and dolphins (26.0 %). In combination with the values obtained from the literature, this result indicates that Carnivora species including Pinnipedia have a much higher capacity of transforming PFOSA to perfluorooctane sulfonic acid (PFOS) than cetacean species. Another notable difference among the species was that the two smaller species (seals and porpoises) with supposedly higher metabolic rates had lower concentrations of the perfluorinated carboxylic acids, which are generally more easily excreted than perfluorinated sulfonamides. Species-specific characteristics should be recognized when PFAS contamination in marine mammals is investigated, for example, several previous studies of PFASs in cetaceans have not quantified PFOSA.