Inhibition kinetics of acid and alkaline phosphatases by atrazine and methomyl pesticides

The main objective of this work was to investigate the kinetic characteristics of acid and alkaline phosphatases isolated from different sources and to study the effects of the herbicide atrazine and insecticide methomyl on the activity and kinetic properties of the enzymes. Acid phosphatase (ACP) was isolated from the tomato plant (Solanum lycopersicum L. var. lycopersicum); alkaline phosphatase (ALP) was isolated from two sources, including mature earthworms (Aporrectodea caliginosa) and larvae of the Egyptian cotton leafworm (Spodoptera littoralis). The specific activities of the enzymes were 33.31, 5.56 and 0.72 mmol substrate hydrolyzed per minute per milligram protein for plant ACP, earthworms ALP and cotton leafworm ALP, respectively. The inhibition kinetics indicated that atrazine and methomyl caused competitive–non-competitive inhibition of the enzymes. The relationships between estimates of K_m and V_max calculated from the Michaelis–Menten equation have been explored. The extent of the inhibition was different, as estimated by the values of the inhibition constant K_i that were found to be 3.34 × 10^?3, 1.12 × 10^?2 and 1.07 × 10^?2 mM for plant ACP, earthworms ALP and cotton leafworm ALP, respectively, with methomyl. In the case of atrazine, K_i were found to be 8.99 × 10^?3, 3.55 × 10^?2 and 1.36 × 10^?2 mM for plant ACP, earthworms ALP and cotton leafworm ALP, respectively.     

Cyp35a2 gene expression is involved in toxicity of fenitrothion in the soil nematode Caenorhabditis elegans

In this study, the effect of organophosphorous (OP) pesticide, fenitrothion (FT), on the non-target organism was investigated using the soil nematode, Caenorhabditis elegans. Toxicity was investigated on multiple biological levels, from organism to molecular levels, such as, immoblity, growth, fertility, development, acetyl cholinesterase (AChE) activity and stress-response gene expressions. FT may provoke serious consequences on the C. elegans population, as it induced significant developmental disturbance. As expected, FT exposure inhibits AChE activity of C. elegans. The increased expression of the cytochrome p450 family protein 35A2 (cyp35a2) gene was also observed in FT exposed worms. To experimentally demonstrate the relationships between organism-level effects and the cyp35a2 gene expression in FT-exposed C. elegans, the integration of the gene expression with biochemical-, and organism level endpoints were attempted using a C. eleganscyp35a2 RNA interference (RNAi) and cyp35a2 mutant (gk317). The 24 h-EC50s of C. elegans on FT exposure were in the order of cyp35a2 RNAi in cyp35a2 mutant (gk317) > cyp35a2 mutant (gk317) > cyp35a2 RNAi in wildtype (N2) > wildtype (N2). The higher EC50 values of cyp35a2 RNAi and cyp35a2 mutant (gk317) compared to that of wildtypeC. elegans strongly supported that cyp35a2 gene plays an important role in the toxicity of FT towards C. elegans. The experiments with cyp35a2 RNAi also indicated that the development disturbance and decreased AChE activity, which were observed in FT exposed wildtype C. elegans were significantly rescued in the cyp35a2 RNAi C. elegans. Overall results suggest that the cyp35a2 may be an important gene for exerting FT toxicity in C. elegans.     

Assessment of molluscicidal activity of essential oils from five Azorean plants against Radix peregra (Müller, 1774)

The molluscicidal activity of essential oils from two endemic (Juniperusbrevifolia; Laurus azorica) and three introduced (Hedychium gardnerianum; Pittosporum undulatum; Psidium cattleianum) Azorean plants against the snail Radix peregra was studied under laboratory conditions. Essential oils from leaves of H. gardnerianum, L. azorica and J.brevifolia presented promising molluscicidal activity on both adults and juveniles stages of R. peregra. The molluscicidal activity of these essential oils was found to be both time and concentration dependent. Lethal concentrations (LC₅₀) varied between 15.4 (L. azorica) and 44.6 ppm (H. gardnerianum) for juveniles and from 45.3 (H. gardnerianum) to 54.6 ppm (J. brevifolia) for R. peregra adults. Ovicidal effect, calculated as percentage of egg hatching, at 100 ppm concentration, was observed in essential oils from P. undulatum flowers (4.2% of hatching) and leaves of H. gardnerianum (4.9%), L. azorica (7.4%) and J. brevifolia (17.7%). The present study is the first attempt to assess the molluscicidal potential of some Azorean plants essential oils against a Lymnaeidae snail. In fact, the H. gardnerianum, L. azorica and J. brevifolia can offer natural alternative tools for the control of R. peregra population, but more research is needed in order to determine the mode of action of these oils and determine the side effects on the ecosystem where this freshwater snail occurs.     

Suitability of different salt marsh plants for petroleum hydrocarbons remediation

The suitability of the salt-marsh species Halimione portulacoides, Scirpus maritimus, Juncus maritimus and an association of the last two for remediation of petroleum hydrocarbons (PHC) in soil was investigated. An outdoor laboratory experiment (microcosm-scale) was carried out using contaminated soil collected in a refinery, as a complement of another study carried out in the refinery environment (mesocosm-scale). Soil samples with old contamination (mainly crude oil) and with a mixture of the old and recent (turbine oil) contamination were tested. Studies in both micro- and mesocosm-scale provided results coherent in substance. The presence of S. maritimus caused removal of old contamination which was refractory to natural attenuation (after 7 months of exposure, efficiency was 13% when only old contamination was present and 40% when the soil also contained recent contamination). H. portulacoides (only included in the microcosm-scale study) revealed also potentiality for PHC remediation, although with less efficiency than S. maritimus. Degradation of recent contamination was also faster in the presence of plants (after 7 months: 100% in the presence of S. maritimus vs. 63% in its absence). As these species are common in salt marsh areas in Atlantic coast of Europe, it is probable they will be also useful for recovering coast sediments. In contrast, J. maritimus and association did not reveal capability to remove PHC from soil, the presence of J. maritimus inhibiting the capability of S. maritimus.     

Molluscicidal activity of Sapindus mukorossi and Terminalia chebula against the freshwater snail Lymnaea acuminata

The molluscicidal activity of Sapindus mukorossi and Terminalia chebula fruit powder against the vector snail Lymnaea acuminata was time and concentration dependent. The molluscicidal activity of T. chebula fruit powder (96 h LC(50):93.59 mg L(-1)) was more pronounced than that of S. mukorossi fruit powder (96 h LC(50):119.57 mg L(-1)). Ethanolic extracts of S. mukorossi and T. chebula fruit powder were more toxic than their other organic solvent extracts. The molluscicidal activity of ethanolic extract of S. mukorossi fruit powder (24h LC(50):2.75 mg L(-1)) was more effective than the ethanolic extract of T. chebula fruit powder (24h LC(50):124.06 mg L(-1)). The 96 h LC(50) of column-purified fraction of S. mukorossi fruit powder was 5.43 mg L(-1) whereas those of T. chebula fruit powder was 7.49 mg L(-1). Column, thin layer and high performance liquid chromatography analysis demonstrates that the active molluscicidal component in S. mukorossi and T. chebula is saponin (96 h LC(50):1.31 mg L(-1)) and tannic acid (96 h LC(50):1.64 mg L(-1)), respectively. These plants may be used as potent source of molluscicides against the snail L. acuminata.     

Biological effects of palytoxin-like compounds from Ostreopsis cf. ovata: A multibiomarkers approach with mussels Mytilus galloprovincialis

Massive blooms of the harmful benthic dinoflagellate Ostreopsis cf. ovata are of growing environmental concern in the Mediterranean, having recently caused adverse effects on benthic invertebrates and also some intoxication episodes to humans.The toxicological potential of produced palytoxin-like compounds was investigated in the present study on a typical marine sentinel species, the mussel Mytilus galloprovincialis. Organisms were sampled during various phases of a O. cf. ovata bloom, in two differently impacted sites. The presence of the algal toxins was indirectly assessed in mussels tissues (mouse test and hemolysis neutralization assay), while biological and toxicological effects were evaluated through the measurement of osmoregulatory and neurotoxic alterations (Na⁺/K⁺-ATPase and acetylcholinesterase activities), oxidative stress responses (antioxidant defences and total oxyradical scavenging capacity), lipid peroxidation processes (level of malondialdehyde), peroxisomal proliferation, organelle dysfunctions (lysosomal membrane stability, accumulation of lipofuscin and neutral lipids), immunological impairment (granulocytes percentage).Obtained results demonstrated a significant accumulation of algal toxins in mussels exposed to O. cf. ovata. These organisms exhibited a marked inhibition of the Na⁺/K⁺-ATPase activity and alterations of immunological, lysosomal and neurotoxic responses. Markers of oxidative stress showed more limited variations suggesting that toxicity of the O. cf. ovata toxins is not primarily mediated by an over production of reactive oxygen species. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess biological alterations and toxicological events associated to blooms of O. cf. ovata in marine organisms.     

Biodegradation of 4-aminobenzenesulfonate by Ralstonia sp. PBA and Hydrogenophaga sp. PBC isolated from textile wastewater treatment plant

A co-culture consisting of Hydrogenophaga sp. PBC and Ralstonia sp. PBA, isolated from textile wastewater treatment plant could tolerate up to 100 mM 4-aminobenzenesulfonate (4-ABS) and utilize it as sole carbon, nitrogen and sulfur source under aerobic condition. The biodegradation of 4-ABS resulted in the release of nitrogen and sulfur in the form of ammonium and sulfate respectively. Ninety-eight percent removal of chemical oxygen demand attributed to 20 mM of 4-ABS in cell-free supernatant could be achieved after 118 h. Effective biodegradation of 4-ABS occurred at pH ranging from 6 to 8. During batch culture with 4-ABS as sole carbon and nitrogen source, the ratio of strain PBA to PBC was dynamic and a critical concentration of strain PBA has to be reached in order to enable effective biodegradation of 4-ABS. Haldane inhibition model was used to fit the degradation rate at different initial concentrations and the parameters μ_max, K_s and K_i were determined to be 0.13 h⁻¹, 1.3 mM and 42 mM respectively. HPLC analyses revealed traced accumulation of 4-sulfocatechol and at least four unidentified metabolites during biodegradation. This is the first study to report on the characterization of 4-ABS-degrading bacterial consortium that was isolated from textile wastewater treatment plant.     

Potential re-colonisation by cladocerans of an acidic tropical pond

To predict how re-colonisation of acidified lakes will proceed, at least two approaches are possible: (i) to compare the life history traits of candidate species and determine which one has the highest fitness, and (ii) to simulate a more realistic scenario carrying out experiments with the grouping of the candidate species, so that the intrinsic rate of natural increase of each species is integrated with its sensitivity to low pH and its ability to compete with the other candidate populations. The objective of the present study was to investigate the future re-colonisation of such acidified systems, taking as case-study a tropical pond (Lagoa das Dunas, Camaçari, BA, Brazil) and four species of cladocerans occurring in nearby water bodies (Ceriodaphniacornuta, Ceriodaphniasilvestrii, Latonopsisaustralis and Macrothrix elegans), by comparing the two above mentioned approaches. The second approach included two sets of in situ microcosms experiments, one simulating the re-colonisation by immigrating ephippia, thus using neonates of each species as colonisers, and another simulating the immigration of adults. Both these simulations followed nearly the same trends. The integration of the effects of a higher temperature, a different photoperiod and species competition determined differences in the species densities ranking between the two approaches: life history versus microcosms. The densities of C.cornuta in the microcosms matched the biphasic concentration/response hormetic model, in the simultaneous presence of two increasingly intense stressors (interspecific competition and acidity), with a low-dose stimulation and a high-dose inhibition. The present study provided, thus, a further support to the acceptance of hormesis in ecotoxicology, also at the population level in multispecies experiments.     

Linking NE1545 gene expression with cell volume changes in Nitrosomonas europaea cells exposed to aromatic hydrocarbons

Nitrosomonas europaea, a model ammonia oxidizing bacterium, was exposed to a wide variety of aromatic hydrocarbons in 3 h batch assays. The expression of NE1545, a phenol sentinel gene involved in fatty acid metabolism, was monitored via quantitative real-time polymerase chain reaction (qRT-PCR) and a Coulter Counter technique was used to monitor changes in cell volume. Decreases in cell volume and NE1545 gene expression correlated strongly with exposure to aromatic hydrocarbons that possessed a single polar group substitution (e.g. phenol and aniline). Aromatic hydrocarbons that contain no polar group substitutions (e.g. toluene) or multiple polar group substitutions (e.g. p-hydroquinone) caused negligible changes in NE1545 expression and cell volume. The oxidation of aromatic hydrocarbons by N. europaea from configurations without a single polar group to one with two polar groups (e.g. p-cresol oxidized to 4-hydroxybenzyl alcohol) and from configurations with no polar groups to one with a single polar group (e.g. ethylbenzene oxidized to 4-ethylphenol) greatly influenced NE1545 gene expression and observed changes in cell volume. Nitrification inhibition in N. europaea by the aromatic hydrocarbons was found to be completely reversible; however, the decreases in cell volume were not reversible suggesting a physical change in cell membrane composition. Ammonia monooxygenase blocking studies showed that the chemical exposure that was responsible for the cell volume decrease and up-regulation in gene expression and not the observed inhibition. N. europaea is the first bacterium shown to experience significant changes in cell volume when exposed to μM concentrations of aromatic hydrocarbons, three orders of magnitude lower than previous studies with other bacteria.     

Comparative bioremediation potential of four rhizospheric microbial species against lindane

Four microbial species (Kocuria rhizophila, Microbacterium resistens, Staphylococcus equorum and Staphylococcus cohnii subspecies urealyticus) were isolated from the rhizospheric zone of selected plants growing in a lindane contaminated environment and acclimatized in lindane spiked media (5-100 μg mL⁻¹). The isolated species were inoculated with soil containing 5, 50 and 100 mg kg⁻¹ of lindane and incubated at room temperature. Soil samples were collected periodically to evaluate the microbial dissipation kinetics, dissipation rate, residual lindane concentration and microbial biomass carbon (MBC). There was a marked difference (p < 0.05) in the MBC content and lindane dissipation rate of microbial isolates cultured in three different lindane concentrations. Further, the dissipation rate tended to decrease with increasing lindane concentrations. After 45 d, the residual lindane concentrations in three different spiked soils were reduced to 0%, 41% and 33%, respectively. Among the four species, S. cohnii subspecies urealyticus exhibited maximum dissipation (41.65 mg kg⁻¹) and can be exploited for the in situ remediation of low to medium level lindane contaminated soils.     

The presence of Bacillus thuringiensis (Bt) protein in earthworms Eisenia fetida has no deleterious effects on their growth and reproduction

Earthworms Eisenia fetida, bred in substances with stover of two genetically-engineered Bacillus thuringiensis (Bt) corns (5422Bt1 (Event Bt11) and 5422CBCL (MON810)) expressing Cry1Ab and their near-isogenic non-Bt corn (5422), were used to investigate the non-target effects of Bt corn on soil-dwelling organisms. Cry1Ab concentrations in substances, casts and guts of E. fetida were also investigated by Enzyme-linked immunosorbent assay (ELISA). More than 90% individuals of E. fetida survived over a period of 30 d, irrespective of whether they received Bt corn or non-Bt corn. Compared to 5422 treatments, significantly higher relative growth rate and more number of new offspring and cocoons of E. fetida were found in 5422Bt1 and 5422CBCL treatments. These results were unlikely to be directly caused by Cry1Ab released from Bt corns but rather by differences in other factors of plants such as plant components (soluble sugar, total organic carbon, total protein and available phosphorus of Bt corns were more than 5422). ELISA results indicated immunoreactive Cry1Ab was detectable in substances, and the casts, guts of E. fetida from Bt corns treatments, of which the highest levels were detected in substances under the corresponding experimental conditions. With the increase of treated time, a strong decline was observed in Cry1Ab from substances and casts of E. fetida, whereas Cry1Ab in guts of E. fetida from 5422Bt1 treatments gradually increased and that from 5422CBCL treatments increased between 14 and 30 d. Therefore, the presence of Cry1Ab in E. fetida had no deleterious effects on their growth and reproduction.     

The exposition of a calcareous Mediterranean soil to toxic concentrations of Cr, Cd and Pb produces changes in the microbiota mainly related to differential metal bioavailability

The involvement of the bacterial community of an agricultural Mediterranean calcareous soil in relation to several heavy metals has been studied in microcosms under controlled laboratory conditions. Soil samples were artificially polluted with Cr(VI), Cd(II) and Pb(II) at concentrations ranging from 0.1 to 5000 mg kg⁻¹ and incubated along 28 d. The lowest concentrations with significant effects in soil respirometry were 10 mg kg⁻¹ Cr and 1000 mg kg⁻¹ Cd and Pb. However, only treatments showing more than 40% inhibition of respirometric activity led to significant changes in bacterial composition, as indicated by PCR-DGGE analyses. Presumable Cr- and Cd-resistant bacteria were detected in polluted microcosms, but development of the microbiota was severely impaired at the highest amendments of both metals. Results also showed that bioavailability is an important factor determining the impact of the heavy metals assayed, and even an inverted potential toxicity ranking could be achieved if their soluble fraction is considered instead of the total concentration. Moreover, multiresistant bacteria were isolated from Cr-polluted soil microcosms, some of them showing the capacity to reduce Cr(VI) concentrations between 26% and 84% of the initial value. Potentially useful strains for bioremediation were related to Arthrobacter crystallopoietes, Stenotrophomonas maltophilia and several species of Bacillus.     

Isolation and characterization of lead-tolerant Ochrobactrum intermedium and its role in enhancing lead accumulation by Eucalyptus camaldulensis

In this study, the potential of rhizospheric bacteria in promoting the growth and Pb accumulation by the woody plant Eucalyptus camaldulensis under hydroponic conditions was investigated for the first time. Three Pb-tolerant bacteria were isolated from the rhizosphere of E. camaldulensis grown in Pb-contaminated soils in the Bo Ngam Pb mine, Thailand. Based on analysis of partial 16S rRNA gene sequence, the three isolates were identified as Microbacterium paraoxydans BN-2, Ochrobactrum intermedium BN-3, and Bacillus fusiformis BN-4. Among these strains, O. intermedium BN-3 showed the highest tolerance to not only Pb but also Cd and Zn. After growth in the presence of Pb, the membranes of O. intermedium BN-3 cells exhibited an increase in unsaturated fatty acid levels but a decrease in fluidity. In hydroponic studies, inoculation of O. intermedium BN-3 significantly increased the biomass and Pb accumulation by E. camaldulensis compared to the uninoculated control. The results suggested the role of the natural rhizospheric bacteria localized to the root surface of E. camaldulensis in promoting Pb accumulation and plant growth. Our results indicate that O. intermedium BN-3 and other indigenous rhizospheric bacteria have the potential to improve the efficiency of phytoremediation of Pb-contaminated sites.     

Toxicity of mixtures of perfluorooctane sulphonic acid with chlorinated chemicals and lipid regulators

The toxicological interaction of perfluorooctane sulphonic acid (PFOS) with the chlorinated pollutants triclosan and 2,4,6-trichlorophenol and the lipid regulators gemfibrozil and bezafibrate was evaluated using the combination index-isobologram equation. The endpoint for bioassays was the growth rate inhibition of the green alga Pseudokirchneriella subcapitata. The results showed that most of the binary combinations assayed exhibited antagonism at all effect levels. The addition of a third component induced a less antagonistic or even synergistic behaviour. This was particularly marked for the ternary mixture of triclosan and 2,4,6-trichlorophenol with PFOS, for which synergism was very strong at all effect levels, with a combination index as low as 0.034 ± 0.002 at EC₅₀ for the mixture. The results obtained indicate that the evaluation of mixture toxicity from single component data using the concentration addition approach could severely underestimate combined toxicity.     

Trophic transfer of pyrene metabolites and nonextractable fraction from Oligochaete (Lumbriculus variegatus) to juvenile brown trout (Salmo trutta)

Cell lines of Etroplus suratensis established in our laboratory were evaluated for their potential use as screening tools for the ecotoxicological assessment of tannery effluent. The cytotoxic effect of tannery effluent in three cell lines derived from eye, kidney and gill tissue of E. suratensis was assessed using multiple endpoints such as Neutral Red (NR) assay, Coomassie Blue (CB) protein assay and Alamar Blue (AB) assay. Acute toxicity tests on fish were conducted by exposing E. suratensis for 96 h to tannery effluent under static conditions. The toxic effect of tannery effluent on the survival of fish was found to be concentration and time dependent. The tannery effluent at the concentration of 15% caused 100% mortality at 96 h whereas the lower concentration (0.5%) caused 13.33% mortality. The cytotoxicity of tannery effluent was found to be similar in the three cell lines tested, independent of the toxic endpoints employed. EC₅₀ values, the effective concentration of tannery effluent resulting in 50% inhibition of cytotoxicity parameters after 48 h exposure to tannery effluent were calculated for eye, kidney and gill cell lines using NR uptake, AB and cell protein assays. Statistical analysis revealed good correlation with r² = 0.95-0.99 for all combinations between endpoints employed. Linear correlations between each in vitro EC₅₀ and the in vivo LC₅₀ data, were highly significant p < 0.001 with r² = 0.977, 0.968 and 0.906 for AB₅₀, NR₅₀, and CB₅₀, respectively.     

Metallothioneins failed to reflect mercury external levels of exposure and bioaccumulation in marine fish--considerations on tissue and species specific responses

The suitability of metallothioneins (MT) in fish as biomarker of exposure to mercury has been questioned. Therefore, this study aimed at investigating the relationship between external levels of exposure, mercury accumulation and MT content, assessing species and tissue specificities. Two ecologically different fish species - Dicentrarchus labrax and Liza aurata - were surveyed in an estuary historically affected by mercury discharges. Total mercury (T-Hg) and MT content were determined in gills, blood, liver, kidney, muscle and brain. All tissues reflected differences in T-Hg accumulation in both species, although D. labrax accumulated higher levels. Regarding MT, D. labrax revealed a depletion in brain MT content and an incapacity to induce MT synthesis in all the other tissues, whereas L. aurata showed the ability to increase MT in liver and muscle. Tissue-specificities were exhibited in the MT inducing potential and in the susceptibility to MT decrease. L. aurata results presented muscle as the most responsive tissue. None of the investigated tissues displayed significant correlations between T-Hg and MT levels. Overall, the applicability of MT content in fish tissues as biomarker of exposure to mercury was uncertain, reporting limitations in reflecting the metal exposure levels and the subsequent accumulation extent.     

Apolipophorin III and transmission electron microscopy as toxicity indicators for harmaline and tea saponin in Spodoptera exigua (Noctuidae: Lepidoptera)

Apolipophorin III, traditionally known for lipid transport in insects is fairly established as toxicity indicator against harmaline and tea saponin during this study. Apolipophorin III expressed in the hemolymph and midgut tissues of 3rd, 4th, 5th larval instars and pupae of Spodoptera exigua. Apolipophorin III presence was further confirmed by achieving its partial cDNA (Genbank accession no. FJ606822) of 448 bp. qRT PCR revealed that tea saponin resulted in significant reduction of gene expression in 3rd and 4th larval instars but increased in 5th instar as compared to control. Harmaline caused gradual increase of gene expression in 3rd, 4th and 5th instars after feeding on the treated diet. Fifth instar larvae synonymously resulted in the highest gene expressions against both the biochemicals. After the injection of harmaline and tea saponin abrupt increase in gene expression of 4th, 5th larval instar and pupae was observed as compared to control treatment. Transmission electron microscopy of midgut epithelium after being fed with harmaline and tea saponin depicted certain cytological changes. Harmaline treatment lead to cytoplasm vacuolization, mitochondrial disruption, spherocrystals with concentric layers, irregular nucleus and floating nuclei in cytoplasm. Tea saponin treatment resulted in denser cytoplasm, higher intracellular osmotic concentration and reduced complement of apical microvilli. Cells were found to have only a few mitochondria and glycogen deposits in comparison to control treatment.     

Subcellular Mn compartation, anatomic and biochemical changes of two grape varieties in response to excess manganese

To explore the underlying mechanism for the high tolerance to excess manganese stress in the grape species (Vitis vinifera Linn), we observed the subcellular compartment of Mn element, anatomic and biochemical responses of two grape cultivars (Combier and Shuijin) under excess Mn stress in semi-controlled environmental condition. Grape species exhibited typical detoxifying or tolerant mechanism as following: first, majority of Mn element accumulated in leaf was excluded into cell wall or comparted into cell vacuole to avoid cellular Mn-toxicity; Mn and other elements were also secreted into leaf surface or deposited in vascular wall; second, only small amount of Mn was located in cellular organ, and excess Mn in chloroplast was detoxified by depositing in starch granule, which serve as a novel detoxifying strategy; additionally, the cellular Mn was further chelated by phytochelatins; third, to quench the toxic oxygen radicals, the total phenolic compounds and polyamine (putrescine and spermidine) were enhanced. Although the obvious symptom of Mn-toxicity was not detected, we observed the dessication symptom under high level of Mn treatment in the two cultivars, such as sunk stomata, thickened palisade tissue, enhanced palisade/spongy tissue ratio and abscisic acid concentration. The growth inhibition and dessication symptom in the two grape cultivars could be largely associated with osmotic stress resulted from high concentration of leaf Mn.     

A rapid bioassay for detecting saxitoxins using a Daphnia acute toxicity test

Bioassays using Daphnia pulex and Moina micrura were designed to detect cyanobacterial neurotoxins in raw water samples. Phytoplankton and cyanotoxins from seston were analyzed during 15 months in a eutrophic reservoir. Effective time to immobilize 50% of the exposed individuals (ET₅₀) was adopted as the endpoint. Paralysis of swimming movements was observed between ∼0.5-3 h of exposure to lake water containing toxic cyanobacteria, followed by an almost complete recovery of the swimming activity within 24 h after being placed in control water. The same effects were observed in bioassays with a saxitoxin-producer strain of Cylindrospermopsis raciborskii isolated from the reservoir. Regression analysis showed significant relationships between ET₅₀vs. cell density, biomass and saxitoxins content, suggesting that the paralysis of Daphnia in lake water samples was caused by saxitoxins found in C. raciborskii. Daphnia bioassay was found to be a sensitive method for detecting fast-acting neurotoxins in natural samples, with important advantages over mouse bioassays.