Establishing a community-wide DNA barcode library as a new tool for arctic research.

DNA sequences offer powerful tools for describing the members and interactions of natural communities. In this study, we establish the to-date most comprehensive library of DNA barcodes for a terrestrial site, including all known macroscopic animals and vascular plants of an intensively studied area of the High Arctic, the Zackenberg Valley in Northeast Greenland. To demonstrate its utility, we apply the library to identify nearly 20 000 arthropod individuals from two Malaise traps, each operated for two summers. Drawing on this material, we estimate the coverage of previous morphology-based species inventories, derive a snapshot of faunal turnover in space and time and describe the abundance and phenology of species in the rapidly changing arctic environment. Overall, 403 terrestrial animal and 160 vascular plant species were recorded by morphology-based techniques. DNA barcodes (CO1) offered high resolution in discriminating among the local animal taxa, with 92% of morphologically distinguishable taxa assigned to unique Barcode Index Numbers (BINs) and 93% to monophyletic clusters. For vascular plants, resolution was lower, with 54% of species forming monophyletic clusters based on barcode regions rbcLa and ITS2. Malaise catches revealed 122 BINs not detected by previous sampling and DNA barcoding. The insect community was dominated by a few highly abundant taxa. Even closely related taxa differed in phenology, emphasizing the need for species-level resolution when describing ongoing shifts in arctic communities and ecosystems. The DNA barcode library now established for Zackenberg offers new scope for such explorations, and for the detailed dissection of interspecific interactions throughout the community.

Effect of a high dose of three antibiotics on the reproduction of a parthenogenetic strain of Folsomia candida (Isotomidae: Collembola).

Folsomia candida Willem (Isotomidae: Collembola) is an edaphic parthenogenetic species commonly used in ecotoxicity studies. We exposed F. candida to a high dose of three antibiotics, tylosin, ampicillin, and oxytetracycline, that target different bacterial groups. Possible toxic effects were assessed through egg production, hatching, and body size. All three antibiotics caused toxic effects. Treatment with oxytetracycline proved the most toxic. This group showed the smallest body size and lowest number of eggs laid, likely the result of a combination of antibiotic toxicity and avoidance of the antibiotic spiked food. Active toxin avoidance by F. candida in toxicological assays may play a role in minimizing their exposure to toxic compounds. Despite the administration of high doses of oxytetracycline, F. candida individuals remained infected with the intracellular bacteria Wolbachia indicating that this strain is resistant to this antibiotic or that the host or its gut flora had detoxified the compound. An increase in percent egg hatch with time was seen in the ampicillin and oxytetracycline treatments, indicating a possible accommodation of the host and/or gut-flora to these antibiotics.

The toxicity testing of double-walled nanotubes-contaminated food to Eisenia veneta earthworms.

Nanotechnology is playing an increasing role in the society, but very little is known about the environmental toxicity of nanotechnological products. Few previous studies have been published with regard to the effect of nanoparticles on soil organisms. The present paper deals with the toxicity of carbon double-walled nanotubes (DWNT) and C60 fullerenes to the soil-dwelling earthworm Eisenia veneta, with the aim of evaluating the lethal and sub-lethal toxicity. Reproduction of E. veneta was affected by DWNT administered through food at concentrations above 37 mg DWNT/kg food. The most sensitive toxicological parameter was reproduction (cocoon production), with no effect on hatchability, survival or mortality at up to 495 mg DWNT/kg and 1000 mg C60/kg.

Nonylphenol stimulates fecundity but not population growth rate (lambda) of Folsomia candida.

The toxicity of nonylphenol (NP) to springtails was pronounced at 40 mg/kg dry weight soil, at which no animals survived. Body length and fecundity were the individual life-history traits significantly stimulated by sublethal concentrations of NP during a 64-day experiment. However, the effects of NP on these traits did not result in a statistically significant increase in population growth rate (lambda). Decomposition analysis indicated that fecundity was the main contributor to the (non-significant) changes observed in lambda. However, since the elasticity of fecundity was very low, large changes in fecundity resulted in a minimal effect on lambda. Juvenile survival had the highest elasticity of all traits, but was not affected by NP, and therefore did not contribute to effects on lambda. This study confirms previous studies showing that effects of chemicals on individual life-history traits are attenuated at the population level and that lambda is an appropriate endpoint for ecotoxicological studies.

Effects and risk assessment of linear alkylbenzene sulfonates in agricultural soil. 3. Sublethal effects on soil invertebrates.

Sewage sludge applied to agricultural soils often contains considerable amounts of linear alkylbenzene sulfonates (LAS). Toxic effects of LAS on soil organisms should, therefore. be evaluated to ensure safe use of sewage sludge as a fertilizer. In this study, dose-response relationships for the toxicity of Na-LAS to six species of soil invertebrates (survival, reproduction, and growth) were established using a sandy, agricultural soil as test substrate. In general, toxic effects on reproduction and growth appeared when the concentration in soil exceeded 40 to 60 mg/kg. Reproduction was approximately fourfold more sensitive in earthworms and enchytracids than in springtails and mites. It is argued that this difference in sensitivity is related to the dependency of soil pore water, which is high in the annelids but comparatively low in the arthropods.

Effects and risk assessment of linear alkylbenzene sulfonates in agricultural soil. 4. The influence of salt speciation, soil type, and sewage sludge on toxicity using the collembolan Folsomia fimetaria and the earthworm Aporrectodea caliginosa as test organisms.

Sewage sludge applied to agricultural soils often contains linear alkylbenzene sulfonates (LAS) in the range of 1 to 10 g/kg dry weight, and their toxicity to relevant soil organisms should, therefore, be assessed to ensure safe use of sewage sludge as a fertilizer. Studies of LAS toxicity to soil organisms are few, and to our knowledge, factors that may influence the toxicity in the field have not been studied in detail. In this paper, we report on the influence of speciation of LAS in the test solution added to soil (soluble Na-LAS vs poorly soluble Ca-LAS or Mg-LAS), the influence of soil type, and the modifying effects of sludge amendment on the toxicity of LAS. These issues were investigated using reproduction of Collembola and growth of juvenile earthworms as test parameters. Speciation of the LAS added to test soil did not have any influence on toxicity for any of the test species. Likewise, in three different agricultural soils (sand, loam, and clay), we found almost equal toxicities. The LAS added to test soil in a sludge-water suspension was equally toxic as when it was added in an aqueous solution. However, anaerobic incubation for 7 and 14 d of the LAS-sludge suspension (with no decay of LAS) caused the toxicity to increase almost threefold in both collembolan and earthworm. The relationships between soil constituents, bioavailability, and toxicity are also discussed.

Effects and risk assessment of linear alkylbenzene sulfonates in agricultural soil. 5. Probabilistic risk assessment of linear alkylbenzene sulfonates in sludge-amended soils.

Linear alkylbenzene sulfonates (LAS) can be found in high concentrations in sewage sludge and, hence, may enter the soil compartment as a result of sludge application. Here, LAS may pose a risk for soil-dwelling organisms. In the present probabilistic risk assessment, statistical extrapolation has been used to assess the risk of LAS to soil ecosystems. By use of a log-normal distribution model, the predicted no-effect concentration (PNEC) was estimated for soil fauna, plants, and a combination of these. Due to the heterogeneous endpoints for microorganisms, including functional as well as structural parameters, the use of sensitivity distributions is not considered to be applicable to this group of organisms, and a direct, expert evaluation of toxicity data was used instead. The soil concentration after sludge application was predicted for a number of scenarios and used as the predicted environmental concentration (PEC) in the risk characterization and calculation of risk quotients (RQ = PEC/PNEC). A LAS concentration of 4.6 mg/kg was used as the current best estimate of PNEC in all RQ calculations. Three levels of LAS contamination (530, 2,600, and 16,100 mg/kg), three half-lives (10, 25, and 40 d), and five different sludge loads (2, 4, 6, 8, and 10 t/ha) were included in the risk scenarios. In Denmark, the initial risk ratio would reach 1.5 in a realistic worst-case consideration. For countries not having similar sludge regulations, the estimated risk ratio may initially be considerably higher. However, even in the most extreme scenarios, the level of LAS is expected to be well beyond the estimated PNEC one year after application. The present risk assessment, therefore, concludes that LAS does not pose a significant risk to fauna, plants, and essential functions of agricultural soils as a result of normal sewage sludge amendment. However, risks have been identified in worst-case scenarios.

Effects of eight polycyclic aromatic compounds on the survival and reproduction of the springtail Folsomia fimetaria L. (Collembola, isotomidae).

The effects of eight polycyclic aromatic compounds on the survival and reproduction of the collembolan Folsomia fimetaria L. were investigated in a well-characterized Danish agricultural soil. With the exception of acridine, polycyclic aromatic hydrocarbons (PAHs) and neutral N-, S-, and O-monosubstituted analogues showed similar toxicities to soil collembolans when the results were expressed in relation to total soil concentrations (mg/kg). The estimated concentrations resulting in a 10% reduction of reproductive output (EC10 values) were based on measured initial concentrations and were for acridine 290 mg/kg, carbazole 10 mg/kg, dibenzofuran 19 mg/kg, dibenzothiophene 7.8 mg/kg, fluoranthene 37 mg/kg, fluorene 7.7 mg/kg, phenantrene 23 mg/kg, and pyrene 10 mg/kg. When the EC10 values were converted to soil pore-water concentrations, they showed a highly significant correlation (r2 = 0.71, p < 0.01) to no-observed-effect concentrations for the freshwater crustacean Daphnia magna, as estimated by a quantitative structure activity relation (QSAR) for baseline toxicity (nonpolar narcosis). Only carbazole and acridine were more than two times more toxic (4.9 and 3.1, respectively) than expected from the Daphnia QSAR data. The latter result indicates that the toxicity of the tested substances is close to that expected for compounds with nonpolar narcosis as the mode of action. However, the relatively large uncertainties in the extrapolation method prevent final conclusions from being drawn.

Effects of di(2-ethylhexyl) phthalate and dibutyl phthalate on the collembolan Folsomia fimetaria.

Lethal and sublethal effects of di(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) on adult individuals of the collembolan Folsomia fimetaria were investigated in the laboratory by the use of small microcosms. Effects of DEHP and DBP were also tested on newly hatched collembolans in a multidish system. The endpoints were juvenile mortality, growth, and development. When exposed to DEHP, adults and juveniles were unaffected at all test concentrations, that is, up to 5,000 mg/kg. However, DBP caused increased adult mortality at 250 mg/kg and juvenile mortality at 25 mg/kg. For DBP, adult reproduction was a more sensitive endpoint than was survival, with an EC10 and EC50 of 14 and 68 mg/kg, respectively. Juvenile molting frequency seems to be a sensitive parameter, because number of cuticles produced by young springtails was reduced at 1 mg/kg. Toxicity was reduced when soil spiked with DBP was stored at 20 degrees C for a period of up to 28 d before adding the animals. Reduction in toxicity of DBP may be due a combination of degradation, evaporation, and adsorption of DBP to soil material. This was confirmed by chemical analyses, which showed a rapid initial disappearance followed by a much slower disappearance. Our results lead to the overall conclusion that significant adverse effects of phthalates on collembolans are not likely to occur as a result of normal sewage sludge application.

Effects of polycyclic aromatic compounds on the drought tolerance of Folsomia fimetaria (Collembola, Isotomidae).

Drought tolerance (survival at 98.2% relative humidity and 20 degress C) was investigated for adult individuals of the springtail Folsomia fimetaria after three weeks of exposure to different polycyclic aromatic compounds (PACs). Seven PACs were investigated (acridine, dibenzofuran, carbazole, fluorene, dibenzothiophene, fluoranthene, and pyrene), with each substance used at several exposure concentrations. Sublethal concentrations of dibenzothiophene, fluorene, fluoranthene, and pyrene caused a dose-related decrease in drought tolerance in exposed adults, whereas no such relationship was found for acridine, dibenzofuran, and carbazole. The organisms used in the drought tolerance study were taken directly from standard ecotoxicity tests that had just been completed, hence the concentrations causing subsequent effects on drought tolerance and those affecting survival and reproduction in the ecotoxicity tests could be compared. Some of the tested substances significantly reduced the drought tolerance of F. fimetaria at concentrations that had little effect on survival, indicating a synergistic effect of the two stressors. However, drought tolerance was affected at concentrations below the threshold value for toxicity in standard tests (no-observed-effect concentration for reproduction) only for dibenzothiophene. In the field, soil organisms exposed to contaminants also face multiple environmental stressors such as drought and cold. Because the reduced drought tolerance is a side effect of chemical exposure, such effects should be considered when extrapolating from laboratory to field conditions.

Effects of the antibiotics oxytetracycline and tylosin on soil fauna.

Antibiotics may enter the terrestrial environment when amending soils with manure. A Note of Guidance on ecological risk assessment of veterinary medicines was issued in January 1998. Hardly any information about ecotoxicological effects of already existing substances are available. This study has tested the effects of two widely used antibiotics, tylosin and oxytetracycline, on three species of soil fauna: Earthworms, springtails and enchytraeids. Neither of the substances had any effect at environmentally relevant concentrations. The lowest observed effect concentration was 3000 mg kg-1 and in many cases no effect was seen even at the highest test concentration of 5000 mg kg-1.

Toxicity of nickel to a soil-dwelling springtail, Folsomia fimetaria (Collembola: Isotomidae).

Exposure of the collembolan Folsomia fimetaria L. to nickel via soil caused significant mortality and reduced growth and reproductive output. Nickel may be present in elevated concentrations due to anthropogenic discharge. Although collembolans are very numerous and important organisms in the soil ecosystem, the effect of nickel has not previously been studied on these organisms. The aim of this study was to investigate the toxic effects of high soil nickel concentrations on the collembolan F. fimetaria following a 3-week exposure in a loamy sand spiked with nickel up to 1000 mg Ni/kg. A 10% decrease in adult female numbers at 427 mg Ni/kg and at 645 mg Ni/kg for adult male numbers was observed for nickel-spiked soil. Juvenile numbers were reduced at 701 mg Ni/kg following a 3-week exposure. The corresponding EC50 values were 786 mg Ni/kg for females, 922 mg Ni/kg for males, and 859 mg Ni/kg for juveniles. The reproductive output seems to be the most sensitive parameter being reduced at soil nickel concentrations above 173 mg Ni/kg (EC10). Adult growth was not affected by soil nickel concentrations up to 1000 mg Ni/kg, but juvenile growth was reduced at concentrations above 480 mg Ni/kg (EC10).

Predator-prey relationships in a two-species toxicity test system.

In a two-species toxicity test system survival and reproduction of both the predator Hypoaspis aculeifer (Gamasida) and the prey Folsomia fimetaria (Collembola) were studied after 21 days of residual exposure to a soil contamination of the insecticide dimethoate. Additional experiments were run to analyze which species-species and compound-species relationships determine the outcome of this two-species experiment. Number of adult F. fimetaria were reduced by both predation and dimethoate exposure, whereas mites preyed less efficiently on adults than on juveniles. At 0.357 mg dimethoate/kg soil, numbers of juvenile F. fimetaria were mainly reduced by predation on adults and juveniles. At 0.7 mg/kg, an additional dimethoate effect was found, which was attributed to an effect on the reproduction of F. fimetaria, mainly due to lethality of adults. It was reasoned that lethal effects on juvenile springtails are less important. Adult H. aculeifer was not affected by dimethoate exposure, whereas numbers of juvenile H. aculeifer demonstrated a decline only at the highest concentration of 0.7 mg/kg. It is hypothesized that this latter effect is possibly due to food depletion caused by a decreased availability of prey, rather than to the lethal effects of dimethoate on juvenile mites. Such a secondary effect of a pesticide application could not have been derived from a single-species toxicity experiment and demonstrates the additional value of a two-species toxicity test system.

Ecological effects assessment of industrial sludge for microarthropods and decomposition in a spruce plantation.

Effects of dried, granulated industrial sludge-containing residues of organic pesticides and precursors were assessed for microarthropod fauna and the decomposition of a spruce forest floor. The investigation was highly realistic, using large plots of about 1/2 ha, and the application was done with professional equipment. The ecological effects of the sludge were compared with the ecological effects of an inorganic fertilizer. Decreases in abundance of the microarthropods ranged from 20 to 80% of the control level after 1 year. Isotoma notabilis Schäffer was the only species that exhibited stimulation at twice the control level due to the sludge. The least affected collembolan species was Lepidocyrtus cyaneus Tullberg, a member of the surface-dwelling life forms. Sensitive species were Isotoma anglicana Lubbock and Isotomiella minor Schäffer. In subhabitats with almost no application of sludge due to a heterogeneous horizontal distribution, the microarthropods were still affected to the same degree as those in the zones of maximum application. Laboratory tests with Folsomia candida Willem gave results similar to the effects on field populations concerning the sludge but revealed no adverse effects of the fertilizer. Decomposition was stimulated to the same extent in the field by the two types of fertilizer but in the laboratory the sludge caused the largest stimulation. The effects on the microarthropod fauna are suggested to be the result of a combination of direct toxicity and changes in the microbial community due to fertilizers.

Osseointegrated implants for single-tooth replacement: a prospective 5-year multicenter study.

One hundred seven Brånemark implants were placed in 92 patients participating in an international multicenter trial on single-implant restorations at seven centers. The patients were followed for 5 years in a prospective study focusing on implant success and crown function. Plaque and gingival indexes, as well as probing depths, were recorded around teeth and implants. The marginal bone level at implants was determined from intraoral radiographs. Only three implants (2.8%) had been lost at the final annual checkup. During the follow-up period, a total of 17 patients dropped out or were excluded because of nonconformity with the protocol. Based on the remaining patients, a total of 86 implants were clinically and radiographically evaluated at the 5-year follow-up period, resulting in a cumulative success rate of 96.6% (71 implants) in the maxillae and 100% (15 implants) in the mandibles. Plaque and gingival indexes showed a similar pattern of good health around both natural teeth and titanium abutments. The marginal bone loss during the 5-year period did not exceed 1 mm as a mean for all implants analyzed. The most frequent complication recorded during the follow-up was loosening of the abutment fixation screw. The outcome of this study indicated that safe and highly predictable results can be obtained for 5 years when Brånemark implants are used to support single-tooth restorations.

Effect of dimethoate on body growth of representatives of the soil living mesofauna.

For the elucidation of effects of a pesticide on life-cycle parameters of arthropods, applicable in the development of ecotoxicological test procedures with soil living animals, attention was drawn to body growth, which has previously been described as a sensitive parameter in Collembola. Body growth of the following three soil-dwelling arthropods was studied under the influence of dimethoate: Folsomia fimetaria (L.), Hypogastrura assimilis Krausbauer, and Hypoaspis aculeifer Canestrini. The effect of this insecticide and acaricide proved to be sex and species specific. Two models were compared for modeling individual body length. The logistic growth model fitted data for all species, whereas the growth model of von Bertalanffy was less applicable for F. fimetaria and H. aculeifer. The growth rate coefficient of the logistic model proved to be a robust parameter useful for predicting effects of dimethoate on juvenile growth of F. fimetaria and H. aculeifer. Adverse effect concentrations on growth for these species were the following: EC10 = 0.11 mg dimethoate/kg dry soil for F. fimetaria and EC10 = 0.59 mg dimethoate/kg dry soil for H. aculeifer. For H. assimilis, no adverse effects on growth were observed at the tested concentrations. The results are compared to other life-cycle parameters (end-points).

The reverse-torque test: a clinical report.

Reverse torque to failure on implants has been studied in animals. Three implants were reverse torqued to failure in a human volunteer, with failure rates between 45 and 58 Ncm. Clinical data are presented on 404 implants examined after reverse-torque testing and loading, with no increase in failure rates. Reverse-torque testing at 20 Ncm appears to be a safe, reliable method for verifying osseointegration with pure titanium screw-shaped implants.

Bending overload and implant fracture: a retrospective clinical analysis.

Thirty-nine patients with implant fractures treated by three of the authors have been analyzed as to probable causes. Thirty-five (90%) of the fractures occurred in the posterior region. Thirty (77%) of the prostheses were supported by one or two implants, which were exposed to a combination of cantilever load magnification and bruxism or heavy occlusal forces. It was concluded that prostheses supported by one or two implants and replacing missing posterior teeth are subjected to an increased risk of bending overload. The literature review indicates that the fracture frequency is low in these situations and this study demonstrates that with appropriate treatment planning, such overload situations can essentially be prevented.