Lead acetate ecotoxicity in tropical soils.

Lead acetate (AcPb) is an important raw material used in chemical industries worldwide. The potential toxicity of AcPb is generally attributed to the presence of Pb. However, the effect of AcPb on the environment as a whole is still poorly known. This study aimed to evaluate AcPb toxicity on three standard species of soil invertebrates and two plant species using ecotoxicology tests. Three tropical soils (Oxisol, Inceptisol, and Tropical Artificial Soil (TAS)) were contaminated with different concentrations of AcPb and one dose of K-acetate (positive control). These soils were used in tests with Eisenia andrei (earthworm), Folsomia candida (springtail), Enchytraeus crypticus (enchytraeid), Zea mays (maize), and Phaseolus vulgaris (common bean). Dose-response curves obtained in the laboratory tests were used to estimate the EC50 values for each species. Among invertebrates, the highest sensitivity to AcPb was observed for E. crypticus in the TAS (EC50 = 29.8 mg AcPb kg-1), whereas for E. andrei and F. candida the highest sensitivity was observed in the Oxisol (EC50 = 141.9 and 1835 mg AcPb kg-1, respectively). Folsomia candida was the least sensitive invertebrate species to AcPb in all soils. Among plant species, Z. mays was less sensitive (EC50 = 1527.5 mg AcPb kg-1) than P. vulgaris (EC50 = 560.5 mg AcPb kg-1) in the Oxisol. The present study evidenced that the toxicity of AcPb should not be attributed uniquely to the presence of Pb, as the treatment containing uniquely Ac provoked the same toxicity as the highest dose of AcPb.

The Self-Assembling Peptide P11-4 Prevents Collagen Proteolysis in Dentin.

The major goal in restorative dentistry is to develop a true regenerative approach that fully recovers hydroxyapatite crystals within the caries lesion. Recently, a rationally designed self-assembling peptide P11-4 (Ace-QQRFEWEFEQQ-NH2) has been developed to enhance remineralization on initial caries lesions, yet its applicability on dentin tissues remains unclear. Thus, the present study investigated the interaction of P11-4 with the organic dentin components as well as the effect of P11-4 on the proteolytic activity, mechanical properties of the bonding interface, and nanoleakage evaluation to artificial caries-affected dentin. Surface plasmon resonance and atomic force microscopy indicated that P11-4 binds to collagen type I fibers, increasing their width from 214 ± 4 nm to 308 ± 5 nm ( P < 0.0001). P11-4 also increased the resistance of collagen type I fibers against the proteolytic activity of collagenases. The immediate treatment of artificial caries-affected dentin with P11-4 enhanced the microtensile bonding strength of the bonding interface ( P < 0.0001), reaching values close to sound dentin and decreasing the proteolytic activity at the hybrid layer; however, such effects decreased after 6 mo of water storage ( P < 0.05). In conclusion, P11-4 interacts with collagen type I, increasing the resistance of collagen fibers to proteolysis, and improves stability of the hybrid layer formed by artificial caries-affected dentin.

Salinization effects on coastal ecosystems: a terrestrial model ecosystem approach.

In coastal areas, intrusion/irrigation with seawater can threaten biodiversity along with crop yields, and the leaching of salts from areas affected by these processes can increase the salinity of water bodies nearby. The aims of this study were to evaluate the effects of salinization on coastal soil ecosystems due to saline intrusion/irrigation. Terrestrial model ecosystems were used to simulate two soil salinization scenarios: (i) seawater intrusion and irrigation with distilled water and (ii) seawater intrusion and irrigation with saline water. Three sampling periods were established: T0-after acclimation period; T1-salinization effects; and T2-populations' recovery. In each sampling period, the abundance of nematodes, enchytraeids, springtails, mites and earthworms, and plant biomass were measured. Immediate negative effects on enchytraeid abundance were detected, especially at the higher level of saltwater via intrusion+irrigation. Eight weeks after the cessation of saline irrigation, the abundance of enchytraeids fully recovered, and some delayed effects were observed in earthworm abundance and plant biomass, especially at the higher soil conductivity level. The observed low capacity of soil to retain salts suggests that, particularly at high soil conductivities, nearby freshwater bodies can also be endangered. Under saline conditions similar to the ones assayed, survival of some soil communities can be threatened, leading to the loss of biodiversity.This article is part of the theme issue 'Salt in freshwaters: causes, ecological consequences and future prospects'.

Toxicity of the bionematicide 1,4-naphthoquinone on non-target soil organisms.

The main goal of the present study was to evaluate the ecotoxicological effects of 1,4-naphthoquinone (1,4-NTQ), a natural-origin compound presenting nematicidal activity, that can be obtained from walnut husk, in plants and soil invertebrates, including non-target soil nematode communities. This research was part of an ongoing project that aims to develop environmentally-friendly nematicides obtained from agricultural residues. The battery of ISO tests included emergence and growth of corn (Zea mays) and rape (Brassica napus); avoidance with the earthworm Eisenia andrei and the collembolan Folsomia candida; and reproduction with the previous species plus the enchytraeid Enchytraeus crypticus. A novel soil nematode community assay was also performed. ISO tests and nematode assays were conducted using a natural uncontaminated soil that was spiked with a range of 1,4-NTQ concentrations. Toxicity of 1,4-NTQ was found for all test-species and the most sensitive were F. candida and E. andrei. After 7 days of exposure to 1,4-NTQ, nematode abundance decreased along the concentration gradient, and a partial recovery was observed after 14 days (1,4-NTQ <48 mg kg-1 soil). The number of nematode families consistently decreased in both periods. Overall, results indicate that a 1,4-NTQ concentration of <20 mg kg-1 could be environmentally safe but preliminary data suggest that it might be ineffective for the target-nematodes, root-knot nematodes, Meloidogyne spp., and root-lesion nematodes, Pratylenchus spp. In addition, if higher dosages of 1,4-NTQ bionematicide are necessary, the potential recovery of non-target organisms under real field scenarios also needs to be assessed.

Recycling organic wastes to agricultural land as a way to improve its quality: A field study to evaluate benefits and risks.

A field study was established to assess the effects of a sewage sludge (SS), a mixed municipal solid waste compost (MMSWC) and a compost produced from agricultural wastes (AWC), in a Vertisol, using Lolium multiflorum L. The amendments were applied for two consecutive years: 6, 12 and 24t dry matter ha-1 for SS, and the amendment doses for MMSWC and AWC were calculated to deliver the same amount of organic matter (OM) per unit area. The amendments had significant beneficial effects on some soil properties (e.g. soil OM, NKjeldahl, extractable P and K), and on plant productivity parameters (e.g. biomass yield, chlorophyll, foliar area). For instance, soil OM increased from 0.78% to 1.71, 2.48 and 2.51%, after two consecutive years of application of 24t dry matter ha-1 of SS, MMSWC and AWC, respectively, while the plant biomass obtained increased from 7.75tha-1 to 152.41, 78.14 and 29.26tha-1, for the same amendments. On the plant, effects were more pronounced for SS than for both compost applications, a consequence of its higher capacity to provide N to the plant in a readily available form. However, after two years of application, the effects on soil properties were more noticeable for both composts, as their OM is more resistant to mineralization, which endures their beneficial effects on soil. Cadmium, Cr, Ni and Pb pseudo-total concentrations, were not affected significantly by the application of the organic wastes to soil, in all tested doses, neither their extractability by 0.01M CaCl2. On the contrary, Cu and Zn pseudo-total concentrations increased significantly in the second year of the experiment, following the application of the higher rate of MMSWC and AWC, although their extractability remained very low (<0.5% of their pseudo-total fraction). Trace elements concentrations in the aboveground plant material were lower than their maximum tolerable levels for cattle, used as an indicator of risk of their entry into the human food chain. Despite these results, it is interesting to note that the SS promoted a significant increase in the foliar concentrations of Cu, Ni and Zn that did not happen in composts application, which can be explained by the reduction of the soil pH, as a consequence of SS degradation in soil. Concluding, if this type of organic wastes were to be used in a single application, the rate could be as high as 12 or even 24tha-1, however, if they are to be applied in an annual basis, the application rates should be lowered to assure their safe application (e.g. to 6tha-1). Moreover, it is advisable to use more stable and mature organic wastes, which have longer lasting positive effects on soil characteristics.

Chemical constituents and their antibacterial activity from the tropical endophytic fungus Diaporthe sp. F2934.

AIMS: To isolate, characterize and determine the antibacterial activities of compounds produced by the endophytic fungus Diaporthe sp. F2934, cultivated on malt extract agar. METHODS AND RESULTS: The fungus was cultivated aseptically in Petri dishes containing malt extract agar at 25°C for 15 days. Crude extract was obtained from mycelium using ethyl acetate and sonication, and was fractioned using classic chromatography and HPLC. The structures of phomosines and chromanones were established by NMR experiments including HMQC, HMBC and COSY. Their molecular formulas were determined by ESI-TOFMS. We obtained six compounds: (1) 4H-1-benzopyra-4-one-2,3-dihydro-5-hydroxy-2,8-dimetyl, (2) 4H-1-benzopyran-4-one-2,3-dihydro-5-hydroxy-8-(hydroxylmethyl)-2-methyl, (3) 4H-1-benzopyra-4-one-2,3-dihydro-5-methoxyl-2,8-dimetyl, (4) phomosine A, (5) phomosine D and (6) phomosine C. Isolated compounds 1, 2 and 5 were inactive against 15 micro-organisms, but phomosines A and C were active against diverse Gram-negative and Gram-positive bacteria. CONCLUSIONS: A group of new chromanones and known phomosines have been isolated from the genus Diaporthe (Diaporthe sp. F2934). The results obtained confirm the wide chemical diversity produced by endophytic fungi, specifically the genus Diaporthe. In addition, phomosines A and C may be considered as antimicrobial agents that can be used to guide the development of new antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Our phylogenetic analysis places Diaporthe sp. F2934 as sister to the Diaporthe cynaroidis clade. Three chromanones were isolated and identified, for the first time, using crude extract obtained from Diaporthe F2934. From this extract phomosines A, C and D were also purified. Regarding Staphylococcus aureus, the inhibition zone diameter (IZD) for phomosine A was 20% higher than the standard drug, vancomycin. When cultivated as described here, Diaporthe sp. F2934 produced new and antimicrobial compounds.

Effects of NaCl and seawater induced salinity on survival and reproduction of three soil invertebrate species.

The increase of global mean temperature is raising serious concerns worldwide due to its potential negative effects such as droughts and melting of glaciers and ice caps leading to sea level rise. Expected impacts on soil compartment include floodings, seawater intrusions and use of saltwater for irrigation, with unknown effects on soil ecosystems and their inhabitants. The present study aimed at evaluating the effects of salinisation on soil ecosystems due to sea level rise. The reproduction and mortality of three standard soil invertebrate species (Folsomia candida, Enchytraeus crypticus, Hypoaspis aculeifer) in standard artificial OECD soil spiked with serial dilutions of seawater/gradient of NaCl were evaluated according to standard guidelines. An increased sensitivity was observed in the following order: H. aculeifer≪E. crypticus≈F. candida consistent with the different exposure pathways: springtails and enchytraeids are exposed by ingestion and contact while mites are mainly exposed by ingestion due to a continuous and thick exoskeleton. Although small differences were observed in the calculated effect electrical conductivity values, seawater and NaCl induced the same overall effects (with a difference in the enchytraeid tests where a higher sensitivity was found in relation to NaCl). The adverse effects described in the present study are observed on soils not considered saline. Therefore, the actual limit to define saline soils (4000 µS cm(-1)) does not reflect the existing knowledge when considering soil fauna.

Tratamiento con láser Nd:YAG en una hemorragia premacular: caso clínico / Premacular hemorrhage treatment with Nd:YAG laser: A clinical case

INTRODUCCIÓN: Presentamos el caso de una paciente con una hemorragia premacular tratada con neodímio (Nd):YAG láser. CASO CLÍNICO: Paciente de 54 años de edad fue a urgencias por pérdida repentina de visión en el ojo derecho (OD) desde el día anterior. La máxima visión corregida del OD era la percepción de la luz y en el fondo de ojo se vio una hemorragia premacular de gran tamaño. Se hizo el tratamiento con Nd:YAG láser en ese día para que drenase la hemorragia hacia la cavidad vítrea. En la hemorragia intravítrea había desaparecido al cabo de un mes y después de 3 meses la visión era 20/25

INTRODUCTION: To report a case of premacular hemorrhage treated with Neodymium (Nd):YAG laser puncture. CASE REPORT: A 54 year-old Caucasian female was seen in the emergency department with complaints of sudden loss of vision in her right eye (RE), detected on the previous day. The best-corrected visual acuity (BCVA) in the RE was reduced to light perception, and fundoscopy evidenced a large and dense premacular hemorrhage. An Nd:YAG laser puncture was performed that day to drain the hemorrhage into the vitreous cavity. The intravitreal hemorrhage cleared in about 1 month, and the BCVA of the RE was 20/25 after 3 months

[Premacular hemorrhage treatment with Nd:YAG laser: a clinical case]. / Tratamiento con láser Nd:YAG en una hemorragia premacular: caso clínico.

INTRODUCTION: To report a case of premacular hemorrhage treated with Neodymium (Nd):YAG laser puncture. CASE REPORT: A 54 year-old Caucasian female was seen in the emergency department with complaints of sudden loss of vision in her right eye (RE), detected on the previous day. The best-corrected visual acuity (BCVA) in the RE was reduced to light perception, and fundoscopy evidenced a large and dense premacular hemorrhage. An Nd:YAG laser puncture was performed that day to drain the hemorrhage into the vitreous cavity. The intravitreal hemorrhage cleared in about 1 month, and the BCVA of the RE was 20/25 after 3 months.

Collagen type IV-related nephropathies in Portugal: pathogenic COL4A5 mutations and clinical characterization of 22 families.

Alport syndrome (AS) is caused by pathogenic mutations in the genes encoding α3, α4 or α5 chains of collagen IV (COL4A3/COL4A4/COL4A5), resulting in hematuria, chronic renal failure (CRF), sensorineural hearing loss (SNHL) and ocular abnormalities. Mutations in the X-linked COL4A5 gene have been identified in 85% of the families (XLAS). In this study, 22 of 60 probands (37%) of unrelated Portuguese families, with clinical diagnosis of AS and no evidence of autosomal inheritance, had pathogenic COL4A5 mutations detected by Sanger sequencing and/or multiplex-ligation probe amplification, of which 12 (57%) are novel. Males had more severe and earlier renal and extrarenal complications, but microscopic hematuria was a constant finding irrespective of gender. Nonsense and splice site mutations, as well as small and large deletions, were associated with younger age of onset of SNHL in males, and with higher risk of CRF and SNHL in females. Pathogenic COL4A3 or COL4A4 mutations were subsequently identified in more than half of the families without a pathogenic mutation in COL4A5. The lower than expected prevalence of XLAS in Portuguese families warrants the use of next-generation sequencing for simultaneous COL4A3/COL4A4/COL4A5 analysis, as first-tier approach to the genetic diagnosis of collagen type IV-related nephropathies.

Salvinorin a and related compounds as therapeutic drugs for psychostimulant-related disorders.

Pharmacological treatments are available for alcohol, nicotine, and opioid dependence, and several drugs for cannabis-related disorders are currently under investigation. On the other hand, psychostimulant abuse and dependence lacks pharmacological treatment. Mesolimbic dopaminergic neurons mediate the motivation to use drugs and drug-induced euphoria, and psychostimulants (cocaine, amphetamine, and methamphetamine) produce their effects in these neurons, which may be modulated by the opioid system. Salvinorin A is a κ-opioid receptor agonist extracted from Salvia divinorum, a hallucinogenic plant used in magico-ritual contexts by Mazateca Indians in México. Salvinorin A and its analogues have demonstrated anti-addiction effects in animal models using psychostimulants by attenuating dopamine release, sensitization, and other neurochemical and behavioral alterations associated with acute and prolonged administration of these drugs. The objective of the present article is to present an overview of the preclinical evidence suggesting anti-addictive effects of salvinorin A and its analogues.

Punctate inner choroidopathy: a clinical case report.

PURPOSE: To report an uncommon case of a 29-year-old Caucasian male diagnosed with unilateral choroidal neovascularization (CNV) secondary to punctate inner choroidopathy leading to visual impairment. METHODS: This is a retrospective and descriptive case report based on data from clinical records, patient observation and analysis of ancillary diagnostic tests. RESULTS: A 29-year-old Caucasian male presented to our emergency department with complaints of decreased central vision in his left eye (LE), detected a few hours before. Best-corrected visual acuity (BCVA) in the LE was counting fingers at 50 cm and BCVA in the right eye was 20/20. Fundoscopy of the LE evidenced multiple round and yellowish lesions in the macula and nasal to the optic nerve, without intraocular inflammation signs. Optical coherence tomography showed increased retinal thickness with detachment of the neuroepithelium and a slight retinal pigment epithelium detachment. Fluorescein angiography revealed hyperfluorescent lesions with blurred borders in the macula. An intravitreal injection of bevacizumab (1.25 mg/0.05 ml) was administered in the LE, which resulted in anatomic and visual improvement. CONCLUSION: We present a rare case of unilateral CNV secondary to punctate inner choroidopathy in a young, myopic male.

Cannabidiol for the treatment of cannabis withdrawal syndrome: a case report.

WHAT IS KNOWN AND OBJECTIVE:   Cannabis withdrawal in heavy users is commonly followed by increased anxiety, insomnia, loss of appetite, migraine, irritability, restlessness and other physical and psychological signs. Tolerance to cannabis and cannabis withdrawal symptoms are believed to be the result of the desensitization of CB1 receptors by THC. CASE SUMMARY:   This report describes the case of a 19-year-old woman with cannabis withdrawal syndrome treated with cannabidiol (CBD) for 10 days. Daily symptom assessments demonstrated the absence of significant withdrawal, anxiety and dissociative symptoms during the treatment. WHAT IS NEW AND CONCLUSION:   CBD can be effective for the treatment of cannabis withdrawal syndrome.

Immunomodulatory/anti-inflammatory effects of Baccharis dracunculifolia leaves.

A possible immunomodulatory/anti-inflammatory effect of Baccharis dracunculifolia (Bd) and its major compound--caffeic acid (Ca)--on cytokines production (IL-1ß, IL-6 and IL-10) by murine macrophages was investigated. Cells were incubated with Bd and Ca, and the inhibitory concentrations were tested before or after macrophages challenge with LPS. Bd and Ca stimulated IL-1ß and inhibited IL-6 and IL-10 production. In LPS-challenge protocols, Bd prevented LPS action either before or after LPS challenge, whereas Ca prevented LPS effects only after LPS addition. Bd modulatory action on cytokines production may be at least in part mediated by Ca, since it has been shown to inhibit the transcription factor NF-κB. Further studies are still needed to evaluate Bd efficacy in inflammatory diseases, in order to explore its antiinflammatory activity in vivo.

Functional and structural parameters to assess the ecological status of a metal contaminated area in the tropics.

Ecological parameters (soil invertebrates, microbial activity, and plant community) were assessed in a metal contaminated site in an abandoned lead smelter and non-contaminated reference sites, as part of an ecological risk assessment (ERA). Vegetation cover inside the smelter area was lower and presented a more homogenous species composition than outside. A more simplified and less abundant vegetation community within the smelter area also simplified the habitat conditions, which in addition to metal toxicity, impaired the soil microbial and faunal communities. A significant reduction in the feeding activity was observed within the smelter area. Also a significant change in community composition of surface dwelling invertebrates was observed at those sites when compared to sites outside the smelter area. Moreover, basal respiration, microbial biomass C, dehydrogenase and phosphatase activity also decreased in several of these points under the smelter area. As a result, a significant impairment of organic material decomposition in the most contaminated sites was observed. Metal contamination affected the ecological status of the site, leading to a risk for ecosystem functioning and provisioning of ecosystem services like organic matter decomposition and nutrient cycling, even 17 years after the end of smelting activities. Regarding the sensitivity of the ecological parameters assessed, most were able to distinguish sites within the smelter area boundaries from those outside. However, only bait lamina (feeding activity), basal respiration and microbial biomass carbon presented high capacity to distinguish the level of soil contamination, since they were significantly correlated with metal loadings, and thus are promising candidates to be integrated in the Ecological Line of Evidence of an ERA.

Pesticide application to agricultural fields: effects on the reproduction and avoidance behaviour of Folsomia candida and Eisenia andrei.

The objective of this work was to assess the impact of pesticide application to non-target soil organisms simulating what happens following pesticide application in agricultural fields and thus obtaining higher realism on results obtained. For that purpose, three commercial formulations containing the insecticides chlorpyrifos and endosulfan and the herbicide glyphosate were applied to a Mediterranean agricultural field. The soil was collected after spraying and dilution series were prepared with untreated soil to determine the impact of the pesticides on the avoidance behaviour and reproduction of the earthworm Eisenia andrei and the collembolan Folsomia candida. A significant avoidance was observed at the recommended field dose in case of endosulfan by earthworms (60 %) and in case of chlorpyrifos by collembolans (64 %). In addition, both insecticides affected the number of juveniles produced by the earthworms (EC(50) were below the recommended field dose). Glyphosate did not seem to affect either earthworms or collembolans in the recommended field dose. Folsomia candida was more sensitive to pesticide application than Eisenia andrei, what was corroborated by the EC(50) and LC(50) values. In conclusion, insecticides may affect the structure of the soil community by reducing the survival of collembolans and the reproductive capacity of collembolans and earthworms.

Short-term changes of metal availability in soil. Part I: comparing sludge-amended with metal-spiked soils.

Sewage sludge application to soils is regulated by its total metal content. However, the real risk of metals is determined by the fraction that is biologically available. The available fraction is highly related to the strength of metal binding by the matrix, which is a dynamic process. The evaluation of the fate of metals in time can contribute increased accuracy of ecological risk assessment. Aiming to evaluate short-term changes in metal availability when metals were applied to soil directly (metal-spiked) or by way of an organic matrix (sludge-amended), a laboratory experiment was performed using open microcosms filled with agricultural soil. A concentration gradient of industrial sludge (11, 15, 55, and 75 t/ha) that was contaminated predominantly with chromium, copper, nickel, and zinc, or soil freshly spiked with the same concentrations of these metals, were applied on top of the agricultural soil. After 0, 3, 6, and 12 weeks, total (HNO(3) 69 %) and 0.01 M CaCl(2)-extractable metal concentrations in soil and metal content in the percolates were measured. Results demonstrated that comparison between sludge-amended and metal-spiked soils may give important information about the role of sludge matrix on metal mobility and availability in soil. In sludge-amended soils, extractable-metal concentrations were independent of the sludge concentration and did not change over time. In metal-spiked soils, metal extractability decreased with time due to ageing and transport of metals to deeper layers. In general, the sludge matrix increased the adsorption of metals, thus decreasing their mobility in soils.

Characterization and validation of a Portuguese natural reference soil to be used as substrate for ecotoxicological purposes.

This study describes the first attempt to validate a Portuguese natural soil (PTRS1) to be used as reference soil for ecotoxicological purposes, aimed to both: (i) obtain ecotoxicological data for the derivation of Soil Screening Values (SSVs) with regional relevance, acting as a substrate to be spiked with ranges of concentrations of the chemicals under evaluation and (ii) act as control and as substrate for the dilution of contaminated soils in ecotoxicological assays performed to evaluate the ecotoxicity of contaminated soils, in tier 2 of risk assessment frameworks, applied to contaminated lands. The PTRS1 is a cambisol from a granitic area integrated in the Central Iberian Zone. After chemical characterization of the soil in terms of pseudo-total metals, PAHs, PCBs and pesticide contents, it was possible to perceive that some metals (Ba, Be, Co, Cr and V) surpass the Dutch Target Values (Dtvs) corrected for the percentage of organic matter and clay of the PTRS1. Nevertheless, these metals displayed total concentrations below the background total concentrations described for Portuguese soils in general. The same was observed for aldrin, endosulfan I, endosulfan II, heptachlor epoxide, and heptachlor; however the Dtvs corrected become negligible. The performance of invertebrate and plant species, commonly used in standard ecotoxicological assays, was not compromised by both soil properties and soil metal contents. The results obtained suggest that the PTRS1 can be used as a natural reference soil in ecotoxicological assays carried out under the scope of ecological risk assessment.

Inhibiting effect of Dorstenia asaroides extracts on cariogenic properties of Streptococcus mutans.

The aim of this study was to examine the effects of Dorstenia asaroides extracts on cariogenic properties of the most cariogenic bacteria, Streptococcus mutans. Hexane (HFr), ethyl-acetate (EFr) and chloroform (CFr) extracts obtained from D. asaroides rhizomes were submitted to chemical analyses, Minimal Inhibitory Concentrations (MIC), glycolysis assay and S. mutans 12-h-old initial biofilms. Chemical characterization showed that all the extracts present furanocoumarins. The MIC values were 80 (HFr and CFr) and 50 µg/mL (EFr). Acid production by S. mutans cells was significantly disrupted by HFr (12.5 mg/mL), EFr (at 2.5; 6.25 and 12.5 mg/mL) and CFr (at 2.5, 6.25 and 12.5 mg/mL) (p < 0.01). Topical applications of HFr, EFr and CFr significantly reduced the colony forming units of S. mutans biofilms compared with those treated with control group in order to 20, 30 and 25% respectively (p < 0.01). The results of the present study suggest that rhizomes of D. asaroides had inhibitory effects on cariogenic properties of S. mutans.

Boric acid as reference substance: pros, cons and standardization.

Boric acid (BA) has been successfully used as reference substance in some standard test guidelines. Due to the fact that previously selected reference substances present a significant risk to human health and/or are banned for environmental reasons, BA is being discussed for broader adoption in OECD or ISO guidelines. To provide input on BA data and contribute to the discussion on its suitability as a reference substance, in the present study BA was tested with two standard soil organisms, Enchytraeus albidus and Folsomia candida, in terms of survival, reproduction and avoidance. Additionally, published data on other organisms was analysed to derive the most sensitive soil dwelling invertebrate (hazard concentration-HC5). Results showed that BA affected the tested organisms, being two times more toxic for collembolans (LC50 = 96; EC50 = 54 mg/kg) than for enchytraeids (LC50 = 325; EC50 = 104 mg/kg). No avoidance behaviour occurred despite the fact that BA affects earthworms. Actually, it is the recommended reference substance for the earthworm avoidance test. Clearly, the suitable performance of BA in one species should not be generalized to other species. Absolute toxicity is not an important criterion for the selection of a reference substance, but it has been proposed that effects should occur within a reasonable range, i.e. <1,000 mg/kg. We could confirm, compiling previous data that for most soil invertebrates, the EC50 is expected to be below 1,000 mg/kg. From these data it could be derived that the most sensitive soil dwelling invertebrate (HC5, 50%) is likely to be affected (EC10) at 28 (8-53) mg H(3)BO(3)/Kg, equivalent to 4.6 (1.4-8.7) mg boron/kg.