INT Toxicity over Natural Bacterial Assemblages from Surface Oligotrophic Waters: Implications for the Assessment of Respiratory Activity.

Plankton community respiration (R) is a major component of the carbon flux in aquatic ecosystems. However, current methods to measure actual respiration from oxygen consumption at relevant spatial scales are not sensitive enough in oligotrophic environments where respiration rates are very low. To overcome this drawback, more sensitive indirect enzymatic approaches are commonly used as R proxies. The in vivo electron transport system (ETSvivo) assay, which measures the reduction of (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride salt, INT) to INT-formazan in the presence of natural substrate levels, was recently proposed as an indirect reliable estimation of R for natural plankton communities. However, under in vivo conditions, formazan salts could be toxic to the cells. Here, we test the toxicity of 0.2 mM of final INT concentration, widely used for ETSvivo assays, on natural bacterial assemblages collected in coastal and oceanic waters off Gran Canaria (Canary Islands, subtropical North Atlantic), in eight independent experiments. After 0.5 h of incubation, a significant but variable decline in cell viability (14-49%) was observed in all samples inoculated with INT. Moreover, INT also inhibited leucine uptake in less than 90 min of incubation. In the light of these results, we argue that enzymatic respiratory rates obtained with the ETSvivo method need to be interpreted with caution to derive R in oceanic regions where bacteria largely contribute to community respiration. Moreover, the variable toxicity on bacterial assemblages observed in our experiments questions the use of a single R/ETSvivo relationship as a universal proxy for regional studies.

Environmental benchmarks based on ecotoxicological assessment with planktonic species might not adequately protect benthic assemblages in lotic systems.

Freshwater ecosystems face widespread diffuse and point-source contamination. Species Sensitivity Distributions (SSDs) have been used as a tool to determine chemical concentration benchmarks that represent protective levels for most species in the environment. Here we used a SSD approach to assess on the adequacy of standard planktonic organisms to reflect the response of benthic communities, critically supporting the structure and function of lotic ecosystems. For the purpose, SSDs reflecting non-lethal responses of standard planktonic and selected benthic organisms were built based on EC50 values (collected in the literature or estimated following testing herein) regarding three model contaminants: potassium dichromate (PD), 3,5-dichlorophenol (DCP) and lead chloride (LC). The derived HC5 estimates were discriminatory between chemicals and the uncertainty associated with the estimate was remarkably low. The HC5 estimates with corresponding uncertainty were generally within the same order of magnitude for the three chemicals tested, with better discrimination between chemicals regarding their hazardous potential being achieved for benthic organisms: DCP was clearly less hazardous than PD, but LC tends to be as hazardous as PD and DCP (assuming the confidence interval ranges). Moreover, benthic communities were more sensitive to both DCP and PD, in this later case the HC5 being lower by more than one order of magnitude than that found for planktonic communities; for LC, confidence intervals overlapped, preventing a feasible assumption regarding differential sensitivity of the compared communities. Microphytobenthos was highlighted as the most sensitive group to the three tested chemicals in SSDs covering the benthic compartment, while SSDs with planktonic organisms did not consistently show trends in sensitivity ordering. Overall, our results suggest that protective benchmarks retrieved from SSDs built with the responses of standard planktonic organisms (which are the most commonly used for regulation purposes) do not adequately protect benthic communities.

Preclinical study of a cost-effective photodynamic therapy protocol for treating oral candidoses.

Photodynamic therapy (PDT) is a promising treatment for oral candidoses. Its use as an alternative to antifungals prevents several adverse effects, including microbial resistance. However, most PDT protocols do not employ devices and consumables commonly available in dental practice, thus influencing treatment affordability. This study aimed to determine the efficacy of a PDT method based on light curing units' blue LEDs combined to a plaque-disclosing composition (5% erythrosine) against C. albicans in culture and in a murine model of oral candidosis. Standard and resistant fungal strains were tested in vitro in planktonic and biofilm forms. PDT (pre-irradiation time periods: 30 and 60 s; irradiation time: 3 min) was compared to control conditions without light and/or erythrosine. Mice with induced oral candidosis (n = 40) randomly received PDT or similar control conditions with subsequent C. albicans count. These mice underwent histological analysis, as well as 12 healthy mice submitted to experimental treatments. PDT completely inactivated C. albicans planktonic cells and biofilm. Control conditions presented minor differences (ANOVA, p < 0.05), with mean values ranging from 5.2 to 6.8 log10 (UFC/mL). Infected mice presented no significant difference in C. albicans counts consequent to treatments (ANOVA, p = 0.721), although the PDT protocol was able to enhance the inflammatory infiltrate in healthy mice. It can be concluded that the tested PDT protocol can inactivate C. albicans but still needs further investigation in order to achieve efficacy and safety.

Assessment of didecyldimethylammonium chloride as a ballast water treatment method.

Ballast water-mediated transfer of aquatic invasive species is considered a major threat to marine biodiversity, marine industry and human health. A ballast water treatment is needed to comply with International Maritime Organization (IMO) ballast water discharge regulations. Didecyldimethylammonium chloride (DDAC) was tested for its applicability as a ballast water treatment method. The treatment of the marine phytoplankton species Tetraselmis suecica, Isochrysis galbana and Chaetoceros calcitrans showed that at 2.5 µL L(-1) DDAC was able to inactivate photosystem II (PSII) efficiency and disintegrate the cells after 5 days of dark incubation. The treatment of natural marine plankton communities with 2.5 µL L(-1) DDAC did not sufficiently decrease zooplankton abundance to comply with the IMO D-2 standard. Bivalve larvae showed the highest resistance to DDAC. PSII efficiency was inactivated within 5 days but phytoplankton cells remained intact. Regrowth occurred within 2 days of incubation in the light. However, untreated phytoplankton exposed to residual DDAC showed delayed cell growth and reduced PSII efficiency, indicating residual DDAC toxicity. Natural marine plankton communities treated with 5 µL L(-1) DDAC showed sufficient disinfection of zooplankton and inactivation of PSII efficiency. Phytoplankton regrowth was not detected after 9 days of light incubation. Bacteria were initially reduced due to the DDAC treatment but regrowth was observed within 5 days of dark incubation. Residual DDAC remained too high after 5 days to be safely discharged. Two neutralization cycles of 50 mg L(-1) bentonite were needed to inactivate residual DDAC upon discharge. The inactivation of residual DDAC may seriously hamper the practical use of DDAC as a ballast water disinfectant.

Assessment of the working range and effect of sodium dichloroisocyanurate on Pseudomonas aeruginosa biofilms and planktonic cells.

Sodium dichloroisocyanurate (NaDCC) is a chemical agent that acts against microorganisms in a manner similar to that of sodium hypochlorite by releasing free available chlorine. NaDCC has been approved by the WHO for the emergency treatment of water and by the US EPA for routine treatment of water. Previous studies assessing the effectiveness of NaDCC for the treatment of water implied that NaDCC should have a wide array of disinfecting effects beyond the treatment of planktonic cells in potable water. In this study the biocidal effects of NaDCC against Pseudomonas aeruginosa cells in different growth modes including planktonic cells and biofilms were explored. The data showed that a 60% dilution of the standard NaDCC solution was effective in the treatment of both P. aeruginosa planktonic cells and biofilms.

Electrochemical disinfection for ballast water management: technology development and risk assessment.

Ballast water is essential in maintaining the balance and structural integrity of ships during voyage. However, it has created biological invasion threats to the ocean environment. An innovative electrochemical technology was developed in this study. The microorganisms regulated by the International Maritime Organization (D2) were used as the target organisms. It was found that the required energy to meet the D2 was below 0.006 kWh/m3. The size of disinfector (m3) was about 0.5% of treatment flow rate (m3/h). The complete disappearance of chlorine in seawater was achieved after three days. The ballast tank corrosion was not worsened due to the application of technology. The ecotoxicity studies showed no toxic effect on fish, invertebrate, and algae. Finally, the environmental risk assessment showed the treated water did not pose threats to the environment. It can therefore be concluded that the technology provides a cost-effective and environmental friendly solution to ballast water management.

The assessment of the antibacterial and antifungal activities of aspirin, EDTA and aspirin-EDTA combination and their effectiveness as antibiofilm agents.

AIMS: To evaluate the antimicrobial activities of aspirin, EDTA and an aspirin-EDTA (A-EDTA) combination against Pseudomonas aeruginosa, Escherichia coli and Candida albicans in planktonic and biofilm cultures. METHODS AND RESULTS: Minimal inhibitory concentrations (MIC) and minimal biocidal concentrations (MBC) were determined using twofold broth microdilution and viable counting methods, respectively. Aspirin's recorded MIC values ranged from 1.2 to 2.7 mg ml(-1). Checkerboard assay demonstrated a synergism in antimicrobial activity upon combination. Aspirin's minimal biofilm eradication concentration values (MBEC) against the established biofilms ranged between 1.35 and 3.83 mg ml(-1). A complete eradication of bacterial biofilms was achieved after a 4-h treatment with the A-EDTA combination. CONCLUSION: Both aspirin and EDTA possess broad-spectrum antimicrobial activity for both planktonic and biofilm cultures. Aspirin used at the MBEC for 24 h was successful in eradicating P. aeruginosa, E. coli and C. albicans biofilms established on abiotic surfaces. Moreover, the exposure to the A-EDTA combination (4 h) effected complete bacterial biofilm eradication. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a continuous need for the discovery of new antimicrobial agents. Aspirin and EDTA are 'nonantibiotic drugs', the combination of which can be used successfully to treat and eradicate biofilms established on abiotic surfaces.

Effects of a herbicide-insecticide mixture in freshwater microcosms: risk assessment and ecological effect chain.

Effects of chronic application of a mixture of the herbicide atrazine and the insecticide lindane were studied in indoor freshwater plankton-dominated microcosms. The macroinvertebrate community was seriously affected at all but the lowest treatment levels, the zooplankton community at the three highest treatment levels, with crustaceans, caddisflies and dipterans being the most sensitive groups. Increased abundance of the phytoplankton taxa Cyclotella sp. was found at the highest treatment level. Threshold levels for lindane, both at population and community level, corresponded well with those reported in the literature. Atrazine produced fewer effects than expected, probably due to decreased grazer stress on the algae as a result of the lindane application. The safety factors set by the Uniform Principles for individual compounds were also found to ensure protection against chronic exposure to a mixture of a herbicide and insecticide at community level, though not always at the population level.

The toxicity of herbicides to non-target aquatic plants and algae: assessment of predictive factors and hazard.

Widely used herbicides sometimes inadvertently contaminate surface waters. In this study we evaluate the toxicity of herbicides to aquatic plants and algae and relate it to environmental herbicide concentrations and exposure scenarios, herbicide formulation and mode of action. This was done experimentally for ten herbicides, using the aquatic macrophyte Lemna minor L. and the green alga Pseudokirchneriella subcapitata (Korshikov) Hindak, supplemented with a database study comprising algae toxicity data for 146 herbicides. The laboratory study showed that herbicide formulations in general did not enhance herbicide efficacy in the aquatic environment. The Roundup formulation of glyphosate proved to be the only exception, decreasing the EC(50) of the technical product for both L. minor and P. subcapitata approximately fourfold. Comparison of the sensitivity of L. minor and P. subcapitata revealed up to 1000-fold higher sensitivity of L. minor for the herbicides categorized as weak acids (pK(a) < 5), emphasizing the importance of higher plants in hazard assessment. Database analyses showed that no herbicide group, categorized by site of action, was significantly more toxic than another. Synthetic auxins were the exception as they are virtually non-toxic to unicellular algae. There was no strong correlation between toxicity to algae and K(ow) of the herbicides, not even within groups having the same site of action. Evaluating all data, few herbicides were toxic at concentrations below 1 microg l(-1), which is the 99.9th percentile of the herbicide concentrations measured in the Danish surveillance programme. Joint action of several herbicides cannot however be excluded.

Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry.

OBJECTIVES: To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures. METHODS: Several CoNS strains were grown planktonically or as biofilms to determine the effect of the mode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic. RESULTS: In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays. CONCLUSIONS: This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis.

Comparing aquatic risk assessment methods for the photosynthesis-inhibiting herbicides metribuzin and metamitron.

Three different risk assessment procedures are described that aim to protect freshwater habitats from risks of the photosynthesis-inhibiting herbicides metribuzin and metamitron. These procedures are (1) the first-tier approach, based on standard toxicity tests and the application of an assessment factor, (2) the Species Sensitivity Distribution (SSD) approach, based on laboratory tests with a wider array of species and the application of a statistical model to calculate the HCx (the Hazardous Concentration for x% of the species), and (3) the model ecosystem approach, based on the evaluation of treatment-related effects in field enclosures. A comparison of the risk assessment procedures reveals that the first-tier approach is the most conservative for metamitron and metribuzin, and that HC5 values (and even HC10 values) based on acute EC50 values of algae and aquatic vascular plants may be used to derive maximum permissible concentrations for single applications. For both compounds these HC5 values were very similar to the ecological threshold concentrations in the enclosure studies. In contrast to model ecosystem experiments, however, HCx values based on lab toxicity tests do not provide information on the recovery potential of sensitive endpoints and on indirect effects, which may be important for regulatory decision-making. In the enclosure study, indirect effects of metribuzin on invertebrate populations were observed at an exposure concentration that was approximately 20 times lower than the corresponding HC5 value based on lab toxicity data for aquatic invertebrates.

Freshwater microcosms-based assessment of eco-toxicological effects of a chemical effluent from the Pilcam industry in Cameroon.

We studied the acute toxicity of a raw effluent from a battery manufacturing plant (Pilcam) in Douala, Cameroon, to a freshwater fish (Oreochromis niloticus), and subsequently evaluated its sub-acute effects on water quality and the biota in freshwater microscosms. The acute toxicity test was based on 96 hrs static renewal bioassays that resulted in 96-h LC50 and LC90 values of 16 and 20.7% (v/v), respectively. The sub-acute experiments were conducted by exposing several species of aquatic organisms (plankton, macroinvertebrates and mollusks) to lower effluent concentrations [1.6%, 8.0%, 16% (v/v)] for six weeks, and monitoring their survival rates, as well as the physical and chemical characteristics of water. These concentrations were based on 10%, 50%, and 100% of the 96 h - median lethal concentrations (LC50) of the effluent to the freshwater fish, Oreochromis niloticus. Significant effects on functional parameters, such as, chlorophyll-a and total protein could not be demonstrated. However, the activity of alkaline phosphatase was significantly inhibited at all concentrations tested. Phytoplankton, zooplankton, macro-invertebrate communities and snails were negatively affected by the effluent application at concentrations ? 8% (v/v), with chlorophyta, ciliates, ostracoda, annelida, planaria and snails being the most sensitive groups. The snails were eliminated after 24 h exposure from microcosms treated with effluent at concentration ? 8% (v/v). Effluent exposure also caused significant effects on water quality parameters (DO, pH, hardness, conductivity, color, turbidity, ammonia) in general at concentrations ? 8% (v/v). Temperature and alkalinity were not significantly affected. Overall, data from this research indicate that a dilution of the Pilcam effluent down to 1.6% does not provide protection against chronic toxicity to aquatic organisms. Further studies are needed to determine the no observable adverse effect level (NOAEL), as well as a chronic reference concentration for this effluent.

Effects of a mixture of two insecticides in freshwater microcosms: II. Responses of plankton and ecological risk assessment.

This paper reports on the chronic effects of a mixture of the insecticides chlorpyrifos and lindane in freshwater microcosms. Chronic treatment levels corresponding to concentrations of 0, 0.005, 0.01, 0.05, 0.1 and 0.5 times the LC50 of the most sensitive standard test organism were evaluated. The zooplankton community structure was altered from the 0.05 * LC50 treatment level upwards. Cladocerans were the most susceptible group, followed by Copepoda and Ostracoda. Rotifera increased in abundance at the higher treatment levels. Increased abundance of some phytoplankton taxa and increased chlorophyll-a levels were found at the two highest treatment levels, most probably a consequence of decreased grazing pressure. Threshold levels for the mixture, both at population and community/ecosystem level, corresponded well with those reported in the literature for the individual compounds. The overall risk assessment indicates no antagonistic or synergistic effects of the mixture at ecosystem level. It was found that the safety factors set by the Uniform Principles for individual compounds also ensure protection against chronic exposure to a mixture of insecticides at community level, though not always at species level.