Ecotoxicological effects of benzoxazinone allelochemicals and their metabolites on aquatic nontarget organisms

Before natural plant allelochemicals can be exploited as biological pesticides against weeds and for disease control, more than the effect on target organisms needs to be known. This study presents results of aquatic biotests using four organisms, namely, a water flea, a freshwater alga, a soil alga, and a luminescent bacterium. The tested substances were 10 benzoxazinone derivatives, 3 of them known to be wheat allelochemicals, benzoxazolin-2(3H)-one (BOA), 6-methoxybenzoxazolin-2(3H)-one (MBOA), and 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one (DIMBOA), and 7 identified degradation intermediates and metabolites. For comparison, two commercial pesticide formulations (BAS, Betanal) were tested by applying the same set of biotests. The data set produced could be seen as an ecotoxicological evaluation for effects of allelochemicals against nontarget organisms and as a base for further risk assessment.     

A comparison of nickel toxicity to pre-exposed earthworms (Eisenia fetida, oligochaeta) in two different test substrates

Earthworms are soil living organisms of high ecological importance. For that reason, earthworms can be considered as feasible biological indicators for many pollutants in soils. Soils are extremely complex and dynamic systems influenced by a number of different abiotic and biological factors determining the effects of potentially toxic substances. To be able to evaluate the toxicity of a single substance to soil living organisms such as earthworms, soil-related influences and interactions need to be excluded. For that purpose, the use of defined, artificial soil free substrates has been widely established. In this study, a new artificial substrate based on the knowledge gained by the application of different methods, in particular the exposure in reconstituted water resembling natural ground water and an agar medium initially developed to void the guts of earthworms, is proposed. Eisenia fetida have been exposed for 96 h to a medium consisting of 1.5% normal melting agarose added to reconstituted ground water. For validation of the substrate, the effects of nickel toxicity, a substance known to be toxic to E. fetida, were evaluated. In addition, to investigate the possible establishment of resistance or adaptation, as well as cross-resistance, E. fetida individuals with a history of previous exposure (more than 10 generations) to nickel and manganese were exposed to different concentrations of nickel. Both media, one consisting of reconstituted ground water [Kiewiet, A.T., Ma, W.-C., 1991. Effect of pH and calcium on lead and cadmium uptake by earthworms in water. Ecotoxicology and Environmental Safety 21, 32-37] and the other of reconstituted ground water plus agar, seemed equally suited for the evaluation of intrinsic effects of nickel and manganese on E. fetida. With regard to the possible development of resistance, E. fetida did not show an increased tolerance towards nickel in spite of being exposed to elevated levels for more than 10 generations; to the contrary, worms with a history of exposure to nickel showed an increased sensitivity towards nickel. No indications of cross-resistance between manganese and nickel pre-exposed worms were detected.     

Einfluß der N-Düngung von Apfelkulturen auf Nitratgehalte im Boden,stickstoffhaltige Reservestoffe im Baum und Ertrag

Effect of N-fertilization in apple orchards on soil nitrate contents, nitrogenous reserves in the tree, and crop yield It should be investigated how nitrate supply affects crop yields and incorporation and mobilization of nitrogenous reserves with Golden Delicious' apple trees on M 7 and M 9, and whether N fertilizer needs are influenced by N reserves in the trees. Two trials were carried out in 1981–1983. In the first one on al loess soil increasing N levels (0, 40, 80, 120 kg N/ha) were applied. In the second one, on a podsolic sandy soil, N was applied either in spring and/or in August (0/0, 80/0, 40/40, 0/80 kg N/ha). N fertilizers were applied to the herbicide strips only. Both orchards were in their best cropping age. Soil nitrate contents were mainly influenced by N-fertilization. Precipitation and drip irrigation caused translocation of nitrate and leaching from the soil layer investigated (0–90 cm). Thus, on the sandy soil almost no nitrate was left in spring, whereas on the loess soil various amounts of nitrate were still available in the subsoil depending on N fertilization in the preciding year. In plots where N fertilization was at optimum, the soil nitrate contents varied between 65 and 130 kg N per hectare herbicide strip over the main growing season (May–August). In the bark N reserves were mainly proteins, but almost equal amounts of protein and soluble N were found in the wood. N reserves were incorporated into the bark mainly between October and December, but even earlier than September in the wood. They were mobilized from April through July. Arginine was stored in the wood in considerably higher amounts than in the bark. Asparagine contents showed a sharp increase immediately after blossom time, particularly in the bark. There was almost no significant influence of the different fertilizer patterns on incorporation and mobilization of nitrogenous compounds. The N-reserves had no effect on crop weight. Highest crop yields were obtained whenever at least 40 kg N/ha were applied in spring. Additional N supply in August did neither increase incorporation of N-reserves nor crop yields. The nitrogenous reserves in the trees can be ignored in estimating N-fertilizer needs. On the other hand, the soil nitrate contents at bud break in April should be taken into account.     

Invertebrates in risk assessment development of a test battery and of short term biotests for ecological risk assessment of soil

4 Conclusion  In general, all biotests used in the ring test are applicable for risk assessment of field soil and contaminated or remediated soil material. Of these, the most sensitive tests were selected as a integral part of the test battery [4]. This selection did not consider either the duration of the tests, which is crucial for the acceptance of tests by the remediation industry, and the effects of pollutants on biomarkers, which indicate the departure of an organism from its normal biochemical or physiological status. Therefore, the proposed test battery has to be supplemented with biotests of short duration. The best choice for a short test using the endpoint reproduction is the biotest withCaenorhabditis elegans which characterises the habitat function of soil. The appropriate screening test for the retention function is the ETr (electron transfer) inhibition test with submitochondrial particles, which provides reliable results in less than 15 min. For screening the habitat function, avoidance tests with soil invertebrates like annelids or enchytraeids are optimal, as both annelids are exposed to the bioavailable pollutants in the soil pore water. The effect of environmental chemicals at the individual level and below can be analysed with the Celegans ToxChip, which gives information of the status of biomarkers on the molecular and biochemical level. Preliminary SOPs for these biotests are formulated. They have still to be evaluated by international ring tests.     

A gamma-glutamyl peptide isolated from onion (Allium cepa L.) by bioassay-guided fractionation inhibits resorption activity of osteoclasts

One gram of onion added to the food of rats inhibits significantly (p < 0.05) bone resorption as assessed by the urinary excretion of tritium released from bone of 9-week-old rats prelabeled with tritiated tetracycline from weeks 1 to 6. To isolate and identify the bone resorption inhibiting compound from onion, onion powder was extracted and the extract fractionated by column chromatography and medium-pressure liquid chromatography. A single active peak was finally obtained by semipreparative high-performance liquid chromatography. The biological activity of the various fractions was tested in vitro on the activity of osteoclasts to form resorption pits on a mineralized substrate. Medium, containing the various fractions or the pure compound, was added to osteoclasts of new-born rats settled on ivory slices. After 24 h of incubation, the tartrate-resistant acid phosphatase positive multinucleated cells, that is, osteoclasts, were counted. Subsequently, the number of resorption pits was determined. Activity was calculated as the ratio of resorption pits/osteoclasts and was compared to a negative control, that is, medium containing 10% fetal bovine serum only and to calcitonin (10(-12) M) as a positive control. Finally, a single peak inhibited osteoclast activity significantly (p < 0.05). The structure of this compound was elucidated with high-performance liquid chromatography-electrospray ionization-mass spectrometry, time-of-flight electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. The single peak was identified as gamma-L-glutamyl-trans-S-1-propenyl-L-cysteine sulfoxide (GPCS). It has a molecular mass of 306 Da and inhibits dose-dependently the resorption activity of osteoclasts, the minimal effective dose being approximately 2 mM. As no other peak displayed inhibitory activity, it likely is responsible for the effect of onion on bone resorption.     

Bioassays for the ecotoxicological and genotoxicological assessment of contaminated soils (Results of a round-robin test)

For the assessment of contaminated or remediated soils, aside from chemical analyses, ecotoxicological tests are performed which focus on the retention function of soils (determined by tests with aqueous soil extracts) and on the habitat function (determined by tests with soil). Whereas numerous tests exist as standardized guidelines for identifying the effect of chemicals, this is not the case for the assessment of soil quality. A round-robin test was performed to monitor the comparability of the results from ecotoxicological test methods on soils and to facilitate the standardization of corresponding test methods. Four contaminated soils were tested using a total number of fifteen test systems, including ecotoxicological and genotoxicological tests with soil extracts and soil. In the second part of this publication series, the results obtained from the tests with soil microorganisms and soil fauna are presented.     

Analysis and detection of the herbicides dimethenamid and flufenacet and their sulfonic and oxanilic acid degradates in natural water

Dimethenamid [2-chloro-N-(2,4-dimethyl-3-thienyl)-N-(2-methoxy-1-methylethyl)acetamide] and flufenacet [N-(4-fluorophenyl)-N-(1-methylethyl)-2-(5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)oxy] were isolated by C-18 solid-phase extraction and separated from their ethanesulfonic acid (ESA) and oxanilic acid (OXA) degradates during their elution using ethyl acetate for the parent compound, followed by methanol for the polar degradates. The parent compounds were detected using gas chromatography-mass spectrometry in selected-ion mode. The ESA and OXA degradates were detected using high-performance liquid chromatography--electrospray mass spectrometry (HPLC-ESPMS) in negative-ion mode. The method detection limits for a 123-mL sample ranged from 0.01 to 0.07 microg/L. These methods are compatible with existing methods and thus allow for analysis of 17 commonly used herbicides and 18 of their degradation compounds with one extraction. In a study of herbicide transport near the mouth of the Mississippi River during 1999 and 2000, dimethenamid and its ESA and OXA degradates were detected in surface water samples during the annual spring flushes. For flufenacet, the only detections at the study site were for the ESA degradates in samples collected at the peak of the herbicide spring flush in 2000. The low frequency of detections in surface water likely is due to dimethenamid and flufenacet being relatively new herbicides. In addition, detectable amounts of the stable degradates have not been detected in ground water.     

Suitability and calibration of a rebound tonometer to measure IOP in rabbit and pig eyes

Objective To utilize the Icare tonometer TAO1 for intraocular pressure (IOP) determination in experimental animals. To calculate true IOP calibration functions for rabbit and porcine eyes. Animals Enucleated eyes of 3‐year‐old healthy experimental rabbits (New Zealand white), and healthy 1 year old experimental pigs (Deutsche Landrasse) were used for the determination of IOP. Procedures Manometric (Geuder GmbH, Heidelberg/Germany) and rebound tonometry (Icare tonometer TAO1, Icare, Helsinki/Finland) were used to record IOP in enucleated animal eyes (rabbit n = 2, pig n = 3). Results The Icare tonometer TAO1 measurements underestimated true IOP by 37–60% in rabbit eyes and 17–63% in porcine eyes. IOP values obtained by both rebound and manometric tonometry for rabbit and porcine eyes followed a linear regression curve. Linear functions were calculated to correct the Icare tonometer TAO1 measurements to true IOP for both rabbit (p = 1.4244p_ic + 4.2421) and porcine eyes (p = 1.0799p_ic + 5.8557). Conclusions The Icare tonometer TAO1 can be utilized for IOP determination in rabbit and porcine eyes when measured values are corrected with the appropriate linear function.     

50 Jahre organische und mineralische Düngung: Humusgehalte,N‐Ausnutzung und N‐Bilanzen

Nach Umbruch einer Naturwiese wurden während 48 Jahren zehn organische und mineralische Düngungsverfahren geprüft. Der Wechsel von der futterbaulichen zur ackerbaulichen Nutzung hatte bei allen Verfahren ein Absinken des Humusgehaltes von 2,3% auf 1,4–2,1% zur Folge. Bei regelmässiger Stallmistdüngung und bei mineralischer Düngung stabilisierte sich der Humusgehalt nach ca. 35 Jahren auf einem neuen Niveau bei etwa 1,4 bis 1,6%. Bei der Düngung mit Klärschlamm oder Torf stellte sich nach 48 Jahren noch kein neues Gleichgewicht ein. Sowohl die ausreichende mineralische wie auch die organische Düngung förderte die Menge und Aktivität der Bodenmikroorganismen. Die pflanzenbauliche Ausnutzung des Stickstoffs (N) von organischen Düngern betrug während der gesamten Versuchsdauer meist nur 10–30%. Die N‐Ausnutzung mineralischer Dünger dagegen stieg von 20–70% auf 50–80%. Dies gibt einen Hinweis auf ein erhöhtes Umweltgefährdungspotential organischer Dünger. Unter Berücksichtigung der abnehmenden Humusgehalte deuten auch die N‐Bilanzen auf eine erhöhte Umweltgefahrdung organischer Düngung.     

Nitrogen turnover of legume seed meals as affected by seed meal texture and quality at different temperatures

The aim was to investigate how legume seed meal texture and corresponding quality affects N turnover at different temperatures. Therefore, the effect of size fractionation ‘fine’ and ‘coarse’ of seed meals of yellow lupin (Lupinus luteus L.), blue lupin (Lupinus angustifolius L.) and faba bean (Vicia faba L.) on net N mineralization and turnover was investigated in an incubation experiment at 5°C, 12°C and 20°C. The differences in N release from the two particle size fractions could not be detected at 12 and 20°C incubation temperature. Moreover, net N mineralization at 5°C was higher during incubation of the coarse particle size fractions than during incubation of the fine fraction. In contrast to the common understanding of temperature dependence of microbial processes, the overall influence of incubation temperature on net N mineralization was less expressed. The formation of microbial biomass was highest at 5°C. The subsequent decrease of soil microbial biomass was only partly reflected by net N mineralization suggesting the formation of microbial residues as a preliminary N sink. The control of the N release from legume seed meals seems to be dominated by the N-immobilizing effects of polyphenols at lower temperatures and of C-rich polymers (hemicelluloses) at higher temperatures.