DE4433384A1 - Toxicological testing for pollutants - Google Patents

Toxicological testing for pollutants

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Publication number
DE4433384A1
DE4433384A1 DE19944433384 DE4433384A DE4433384A1 DE 4433384 A1 DE4433384 A1 DE 4433384A1 DE 19944433384 DE19944433384 DE 19944433384 DE 4433384 A DE4433384 A DE 4433384A DE 4433384 A1 DE4433384 A1 DE 4433384A1
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Prior art keywords
living material
protein
test
nitrogen
pollutants
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Granted
Application number
DE19944433384
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German (de)
Other versions
DE4433384C2 (en
Inventor
Klaus Dr Jung
Irmgard Sobotta
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Helmholtz Zentrum fuer Umweltforschung GmbH UFZ
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Helmholtz Zentrum fuer Umweltforschung GmbH UFZ
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Priority to DE19944433384 priority Critical patent/DE4433384C2/en
Publication of DE4433384A1 publication Critical patent/DE4433384A1/en
Application granted granted Critical
Publication of DE4433384C2 publication Critical patent/DE4433384C2/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity

Abstract

Toxicological test to determine the effect of a pollutant on nitrogen metabolism and protein synthesis in living material, namely meristematic plant tissue (epicotyl sections or root tips), microorganisms or animal cells or tissues, comprises incubating the living material with a <15>N-labelled nitrogen source (nitrate, ammonium or amino acid) in the presence and absence of the pollutant under standardised conditions for a fixed time, measuring the relative abundance (RA) of <15>N by isotope analysis of the living material or a protein fraction of the material, and calculating the RA(test)/RA(control) quotient.

Description

Die Erfindung betrifft die Testung ökotoxikologischer Wirkungen bestimmter Substanzen bzw. Substanzgemische auf biologische Systeme.The invention relates to ecotoxicological testing Effects of certain substances or mixtures of substances on biological systems.

Die Ökotoxikologie beschäftigt sich mit der Umweltge­ fährlichkeit von Stoffen. Sie verarbeitet Erkenntnisse aus der Biologie (Ökologie), Medizin (Toxikologie) und der Rechtswissenschaft (Umweltrecht) und untersucht die Wirkungen von Stoffen auf Organismen.Ecotoxicology is concerned with the environment dangerousness of substances. It processes knowledge from biology (ecology), medicine (toxicology) and of law (environmental law) and examines the Effects of substances on organisms.

Bekannte ökotoxikologische Untersuchungen bedienen sich des Einzelspezies-Tests im Labor, beispielsweise mit Algen (vgl. F. Korte, Lehrbuch der ökologischen Chemie, Georg-Thieme-Verlag, Stuttgart-New York 1987, S. 215). Die Beeinflussung der Algen durch die Teststoffe wird durch Meßparameter, wie die Wachstumsreduktion oder physiologische Veränderungen ermittelt.Known ecotoxicological studies make use of it of the single species test in the laboratory, for example with Algae (see F. Korte, textbook of ecological chemistry, Georg-Thieme-Verlag, Stuttgart-New York 1987, p. 215). The algae are influenced by the test substances through measurement parameters such as growth reduction or physiological changes determined.

Nachteile bekannter ökotoxikologischer Untersuchungen bestehen insbesondere im Zeitaufwand, der erforderlich ist, um eine Aussage zu treffen, sowie darin, daß Wachstumsreduktion oder Veränderungen erst dann sicht­ bar werden, wenn das Lebendmaterial nachhaltig geschä­ digt ist.Disadvantages of known ecotoxicological studies consist in particular in the time required is to make a statement as well as in that Only then will growth slowdown or change become cash if the living material is sustainably damaged is due.

Ebenso werden Rädertierchen (vgl. F. Korte, Lehrbuch der ökologischen Chemie, Georg-Thieme-Verlag, Stuttgart-New York 1987, S. 216) und Fische in Tests verwendet.Also rotifers (see F. Korte, textbook ecological chemistry, Georg-Thieme-Verlag, Stuttgart-New York 1987, p. 216) and fish in tests used.

Die Anwendung stabiler Isotope in der Pflanzenphysio­ logie und Biochemie sowie in der ¹⁵N-Tracertechnik und ¹⁵N-Isotopenanalytik ist an sich bekannt.The use of stable isotopes in plant physiology and biochemistry as well as in ¹ N tracer technology and ¹⁵N isotope analysis is known per se.

Das Ziel der Erfindung besteht darin, ökötoxikologische Wirkungen von Substanzen oder Substanzgemischen auf pflanzliche Systeme in kurzer Zeit zu bestimmen und daraus Aussagen zum ökotoxikologischen Potential abzuleiten.The aim of the invention is eco-toxicological Effects of substances or mixtures of substances on to determine herbal systems in a short time and from this statements on the ecotoxicological potential to derive.

Erfindungsgemäß wird die Aufgabe dadurch gelöst, daß die Stickstoff- Inkorporation/Proteinsyntheseleistung pflanzlicher Gewebe unter Schadstoffeinfluß durch Einsatz des stabilen, nicht radioaktiven Stickstoffiso­ tops ¹⁵N bestimmt wird und durch einen Vergleichstest mit schadstofffreiem pflanzlichem Gewebe die Störungen, die Schadstoffe (Xenobiotika) im pflanzlichen Stickstoff- bzw. Proteinstoffwechsel bewirken, festgestellt werden.According to the invention the object is achieved in that nitrogen incorporation / protein synthesis performance vegetable tissue under the influence of pollutants  Use of the stable, non-radioactive nitrogen iso tops ¹⁵N is determined and by a comparative test with pollutant-free vegetable tissue the disorders, the pollutants (xenobiotics) in the plant Cause nitrogen or protein metabolism, be determined.

Durch gleichzeitige Applikation ¹⁵N-markierter anor­ ganischer (Nitrat, Ammonium) oder organischer (Aminosäuren) Stickstoff-Quellen als Tracer und wasserlöslicher Xenobiotika an meristematische Pflanzengewebe (Epikotylsektion, Wurzelspitzen) oder Mikroorganismen oder tierische Zellen oder Gewebe gelingt es, nach einer definierten Inkubationszeit unter standardisierten Bedingungen über die ¹⁵N-Iso­ topenanalyse des pflanzlichen Gewebes bzw. relevanter N- bzw. Proteinfraktionen (löslicher N, lösliches Protein, Gesamtprotein) im Vergleich zu den unbe­ handelten Kontrollgeweben die metabolische Aktivi­ tät/Proteinsyntheseleistung des Pflanzengewebes oder anderen Lebendmaterials unter Schadstoffeinwirkung zu bestimmen.By simultaneous application of ¹⁵N-labeled anor ganic (nitrate, ammonium) or organic (Amino acids) nitrogen sources as tracers and water-soluble xenobiotics to meristematic Plant tissue (epicot section, root tips) or Microorganisms or animal cells or tissues succeeds after a defined incubation period under standardized conditions via the ¹⁵N iso top analysis of plant tissue or more relevant N or protein fractions (soluble N, soluble Protein, total protein) compared to the unbe acted control tissues the metabolic activi activity / protein synthesis performance of plant tissue or other living material under the influence of pollutants determine.

Hierzu wird der Quotient QW gebildetThe quotient Q W is formed for this

Hohe Quotienten (QW ∼ 1.0) bedeuten, daß keine oder nur eine geringe Beeinflussung der pflanzlichen Stickstoff-Stoffwechselkapazität/Proteinsynthese­ leistung durch das Xenobiotikum verursacht wird, re­ spektive das ökotoxikologische Potential der betreffen­ den Substanz bzw. des Substanzgemisches (Abwasser, De­ poniesickerwässer) gering ist. Umgekehrt gestatten niedrige Quotienten differenzierte Bewertungen des ökotoxikologischen Potentials.High quotients (Q W ∼ 1.0) mean that there is little or no influence on the plant nitrogen metabolism / protein synthesis performance by the xenobiotic; is low. Conversely, low quotients allow differentiated assessments of the ecotoxicological potential.

Entsprechend den Richtlinien der Internationalen Atom­ energie-Organisation ist die Anreicherung (enrichment) stabiler Isotope die Modifikation der natürlichen Iso­ topenverhältnisse in Richtung eines (gewöhnlich des selteneren) stabilen Isotops.According to the guidelines of the International Atom Energy organization is enrichment stable isotopes the modification of the natural iso top relationships towards a (usually the rarer) stable isotope.

Die Anreicherung wird angegeben als Häufigkeit (abun­ dance) in Atom-% (At.-%) bzw. Exzeßhäufigkeit (At.-% exc.). Dies ist die Häufigkeit eines stabilen Isotops minus der natürlichen Häufigkeit von ∼ 0,37 At.-%.Enrichment is given as frequency (abun dance) in atomic% (At .-%) or excess frequency (At .-% exc.). This is the frequency of a stable isotope minus the natural frequency of ∼ 0.37 at .-%.

Beispiel: Stickstoff mit 10.0 At.-% ¹⁵N ist mit ¹⁵N relativ angereichert und enthält 9.63 At.-% ¹⁵N excess. Der Test ist geeignet, da nicht Photosynthese-aktive pflanzliche Gewebe eingesetzt werden, potentiell Tierversuche zu ersetzen.Example: Nitrogen with 10.0 at% ¹⁵N is with ¹⁵N relatively enriched and contains 9.63 at .-% ¹⁵N excess. The test is suitable because it is not photosynthesis-active Vegetable tissues are used, potentially To replace animal testing.

Vorteilhaft ist es, für den Test als Lebendmaterial Pflanzenteile zu verwenden.It is advantageous for the test as a living material Use plant parts.

Besonders vorteilhaft ist es, für den Test als Lebend­ material Pflanzenteile von Erbsen (Pisum arvense) zu verwenden, wodurch Tierversuche substituiert werden können. It is particularly advantageous for the test as living material plant parts of peas (Pisum arvense) too use, whereby animal experiments can be substituted.  

Die Erfindung wird zusammenfassend durch das folgende Schema erläutert. Der Test wird mit dem Namen Ecotoxicological Stable Isotope Metabolic Assay (ESIMA) bezeichnet.The invention is summarized by the following Scheme explained. The test is named Ecotoxicological Stable Isotope Metabolic Assay (ESIMA) designated.

Applikation des Xenobiotikums und des ¹⁵N-Tracers in einem Inkubationsmedium an das pflanzliche Gewebe bzw. biologische System Application of the xenobiotic and the ¹⁵N tracer in an incubation medium to the plant tissue or biological system

Die Erfindung ist geeignet für die Bestimmung des ökotoxikologischen Potentials vonThe invention is suitable for determining the ecotoxicological potential of

  • - organischen Substanzen- organic substances
  • - anorganischen Substanzen- inorganic substances
  • - Substanzgemischen unterschiedlicher Herkunft (Kombinationswirkungen)
    • - Extrakte biologischer Probematerialien
    • - Bodenextrakte
    • - Deponiesickerwässer, Abwässer
    • - schadstoffbelastete wäßrige Medien
    - substance mixtures of different origins (combination effects)
    • - Extracts of biological sample materials
    • - soil extracts
    • - landfill leachate, waste water
    • - polluted aqueous media

in kürzester Zeit und mit hoher Genauigkeit. Durch Kombination des Tests mit organischen und anor­ ganischen Analyseverfahren sind über das Testmaterial Aussagen zur Bioakkumulation und Bioverfügbarkeit von Xenobiotika sowie deren biologische Wirkung möglich.in the shortest possible time and with high accuracy. By combining the test with organic and anor ganic analysis methods are about the test material Statements on the bioaccumulation and bioavailability of Xenobiotics and their biological effects possible.

Die Erfindung soll anschließend an zwei Beispielen erläutert werden.The invention will then be based on two examples are explained.

Testmaterial:
Epikotylsektionen von Erbsen (Pisum arvense)Test material:
Epicotyl Sections of Peas (Pisum arvense)

Anzucht:
Erbsen für vier Tage einquellen und im Dunklen bei 25°C zum Keimen bringen. Ein Zentimeter lange Gewebsabschnitte inkubieren.Cultivation:
Soak peas for four days and germinate in the dark at 25 ° C. Incubate one centimeter long sections of tissue.

Inkubationsmedium:
1% Saccharose
0,05 Vol.-% Tween 20 (Emulgator) mol Penicillin (Langzeit-Test)
0,1% (¹⁵N) Ammoniumchlorid (Tracer)Incubation medium:
1% sucrose
0.05 vol.% Tween 20 (emulsifier) mol penicillin (long-term test)
0.1% (¹⁵N) ammonium chloride (tracer)

Testsubstanz /SubstanzgemischTest substance / substance mixture

Inkubationsbedingungen:
Schüttelansatz mit 20 Gewebesektionen bei 25°C im Dunkeln. Incubation conditions:
Shake with 20 tissue sections at 25 ° C in the dark.

Aufarbeitung:
Analyse der gewaschenen Gewebsabschnitte direkt oder nach Extraktion der relevanten Stickstoff-Fraktion (Protein nach Homogenisation der inkubierten Gewebe in 80% Äthanol und Präzipitation bei 4°C). Überführen des biologischen Materials in ein Ammoniumsalz durch Aufschluß mit konz. Schwefelsäure und Wasser­ dampfdestillation (Kjeldahl).Refurbishment:
Analysis of the washed tissue sections directly or after extraction of the relevant nitrogen fraction (protein after homogenization of the incubated tissue in 80% ethanol and precipitation at 4 ° C.). Transfer of the biological material into an ammonium salt by digestion with conc. Sulfuric acid and steam distillation (Kjeldahl).

Isotopenanalyse:
Bestimmung der ¹⁵N Exzeßhäufigkeit durch Massenspek­ trometrie oder Emissionsspektrometrie (¹⁵N Isotopenana­ lyse ausgehend vom Ammoniumsalz).Isotope analysis:
Determination of the ¹⁵N excess frequency by mass spectrometry or emission spectrometry (¹⁵N isotopic analysis starting from the ammonium salt).

Aufgabe: Bestimmung der Wirkstoffinkorporation in Pisum-Epikotylgewebe unter Einwirkung des Herbicides MCPA unterschiedlicher Konzentration und des Peptidyl­ transferase-Inhibitors Cycloheximids nach 5-stündiger Inkubation.Task: Determination of the active ingredient incorporation in Pisum epicotyl tissue under the influence of herbicides MCPA of different concentrations and peptidyl transferase inhibitors cycloheximids after 5 hours Incubation.

Testfrage: Proteinsyntheseleistung
Testmaterial: Pisum arvense (Epikotyle)
Tracer: [¹⁵N]-Ammoniumchlorid: 96,2 At.-% ¹⁵N
Testsubstanz: MCPA, Cycloheximid
Inkubation: Temp.: 25°C, Dauer: 5 Std.Test question: protein synthesis performance
Test material: Pisum arvense (epicotyle)
Tracer: [¹⁵N] ammonium chloride: 96.2 at% ¹⁵N
Test substance: MCPA, cycloheximide
Incubation: Temp .: 25 ° C, duration: 5 hours

Bemerkungen: Remarks:  

Die Testergebnisse zeigen, daß ausgehend von einfachen, kostengünstigen, anorganischen Tracersubstanzen (¹⁵NH₄Cl) allein über die Bestimmung der ¹⁵N-Isotopen­ häufigkeiten des pflanzlichen Materials (Pisum- Epikotylsektionen) sehr rasch und sensitiv eine Beein­ flussung des N- und Proteinstoffwechsel durch Schad­ stoffe nachgewiesen werden kann. Dies ermöglicht eine differenzierte Bewertung des ökotoxikologischen Poten­ tials von Schadstoffen und Schadstoffgemischen sowie die Ableitung von prophylaktischen und sanierenden Maßnahmen.The test results show that starting from simple, inexpensive, inorganic tracer substances (¹⁵NH₄Cl) solely by determining the ¹⁵N isotopes frequencies of the plant material (pisum Epicot section) very quickly and sensitively one leg Flow of N and protein metabolism through harm substances can be detected. This enables one differentiated assessment of the ecotoxicological potential tials of pollutants and pollutant mixtures as well the derivation of prophylactic and remedial Activities.

Claims (3)

1. Ökotoxikologischer Stoffwechseltest, dadurch ge­ kennzeichnet, daß die Stickstoffstoffwechselkapazität/ Proteinsyntheseleistung lebenden Materials unter Schadstoffeinfluß durch Einsatz des stabilen, nicht radioaktiven Stickstoffisotops ¹⁵N bestimmt wird und durch einen Vergleichstest mit schadstofffreiem Lebendmaterial die Störungen, die Schadstoffe (Xenobiotika) im Stickstoff- bzw. Proteinstoffwechsel des Lebendmaterials bewirken, festgestellt werden.
Durch gleichzeitige Applikation ¹⁵N-markierter anor­ ganischer (Nitrat, Ammonium) oder organischer (Aminosäuren) Stickstoff-Quellen als Tracer und wasserlöslicher Xenobiotika an meristematische Pflanzengewebe (Epikotylsektion, Wurzelspitzen) oder Mikroorganismen oder tierische Zellen oder Gewebe gelingt es, nach einer definierten Inkubationszeit unter standardisierten Bedingungen über die ¹⁵N-Iso­ topenanalyse des pflanzlichen Gewebes bzw. relevanter N- bzw. Proteinfraktionen (löslicher N, lösliches Protein, Gesamtprotein) im Vergleich zu den unbe­ handelten Kontrollgeweben die metabolische Aktivität/Proteinsyntheseleistung des Pflanzengewebes oder anderen Lebendmaterials unter Schadstoffeinwirkung zu bestimmen.
Hierzu wird der Quotient QW gebildet 1. Ecotoxicological metabolism test, characterized in that the nitrogen metabolism capacity / protein synthesis performance of living material under the influence of pollutants is determined by using the stable, non-radioactive nitrogen isotope ¹⁵N and by a comparative test with pollutant-free living material, the disturbances, the pollutants (xenobiotics) in the nitrogen or protein metabolism of the living material can be determined.
Simultaneous application of ¹⁵N-labeled inorganic (nitrate, ammonium) or organic (amino acids) nitrogen sources as tracers and water-soluble xenobiotics to meristematic plant tissue (epicotyl section, root tips) or microorganisms or animal cells or tissue succeeds after a defined incubation period under standardized To determine the metabolic activity / protein synthesis performance of the plant tissue or other living material under the influence of pollutants by means of the ¹ -N isotope analysis of the plant tissue or relevant N or protein fractions (soluble N, soluble protein, total protein) in comparison to the untreated control tissues.
The quotient Q W is formed for this 2. Test nach Anspruch 1, dadurch gekennzeichnet, daß als Lebendmaterial Pflanzenteile verwendet werden.2. Test according to claim 1, characterized in that as Living material plant parts can be used. 3. Test nach Anspruch 1 und 2, dadurch gekennzeichnet, daß als Lebendmaterial Pflanzenteile von Erbsen (Pisum arvense) verwendet werden.3. Test according to claim 1 and 2, characterized in that plant parts of peas (Pisum arvense) can be used.
DE19944433384 1994-09-09 1994-09-09 Ecotoxicological metabolism test with stable isotopes Expired - Fee Related DE4433384C2 (en)

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Cited By (4)

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WO1999007876A2 (en) * 1997-08-12 1999-02-18 Byk Gulden Lomberg Chemische Fabrik Gmbh Method for assaying cellular protein
WO2000013025A1 (en) * 1998-08-31 2000-03-09 University Of Washington Stable isotope metabolic labeling for analysis of biopolymers
WO2001036963A2 (en) * 1999-11-18 2001-05-25 Gsf-Forschungszentrum Fur Umwelt Und Gesundheit Gmbh Method for detecting noxious substances using plant cell suspensions
EP2293056A3 (en) * 1999-04-20 2011-06-29 Target Discovery, Inc. A method for analysing a metabolic pathway

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DE102013223891B4 (en) * 2013-11-22 2015-09-17 Joanneum Research Forschungsgesellschaft Mbh Cartridge disposal collectors and method for entry measurement of atmospheric substances

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999007876A2 (en) * 1997-08-12 1999-02-18 Byk Gulden Lomberg Chemische Fabrik Gmbh Method for assaying cellular protein
WO1999007876A3 (en) * 1997-08-12 1999-04-29 Byk Gulden Lomberg Chem Fab Method for assaying cellular protein
WO2000013025A1 (en) * 1998-08-31 2000-03-09 University Of Washington Stable isotope metabolic labeling for analysis of biopolymers
EP2293056A3 (en) * 1999-04-20 2011-06-29 Target Discovery, Inc. A method for analysing a metabolic pathway
WO2001036963A2 (en) * 1999-11-18 2001-05-25 Gsf-Forschungszentrum Fur Umwelt Und Gesundheit Gmbh Method for detecting noxious substances using plant cell suspensions
WO2001036963A3 (en) * 1999-11-18 2002-04-04 Gsf Forschungszentrum Umwelt Method for detecting noxious substances using plant cell suspensions

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