natgillin/translations-raw huggingface dataset:
En kontrolserie indeholdende kvartssand (2,5 g pr. beholder) bør testes for kemikalier, der kræver administration i overensstemmelse med fremgangsmåden i punkt 21.
Examples for appropriate growth conditions for certain crop species
Testtemperaturen er 20 °C ± 2 °C.
The following conditions have been found suitable for 10 crop species, and can be used as a guidance for tests in growth chambers with certain other species as well:
For at sikre at ormene ikke forlader jorden, udføres testen under kontrollerede lys-/mørkecyklusser (helst 16 timers lys og 8 timers mørke) med belysning på 400-800 lux i testbeholdernes område.
Carbon dioxide concentration:
For at kontrollere jordfugtigheden vejes beholderne i begyndelsen af testen og efterfølgende en gang om ugen.
Relative humidity:
Vægttab erstattes ved tilsætning af en passende mængde deioniseret vand.
70 ± 5 % during light periods and 90 ± 5 % during dark periods;
Det skal bemærkes, at tab af vand kan reduceres ved at opretholde en høj luftfugtighed (> 80 %) i testinkubatoren.
Temperature:
Fugtindholdet og pH måles i begyndelsen og slutningen af både den indledende test og den endelige test.
25 ± 3 °C during the day, 20 ± 3 °C during the night;
Målinger foretages i kontrollen og behandlede (alle koncentrationer) jordprøver, der er klargjort og opbevaret på samme måde som testkulturerne, men uden orme.
Photoperiod:
Foder tilsættes kun til disse jordprøver i begyndelsen af testen for at fremme mikrobiel aktivitet.
16 hour light/8 hour darkness, assuming an average wavelength of 400 to 700 nm;
Den tilsatte mængde foder skal være den samme som den, der tilsættes til testkulturerne.
Light:
Det er ikke nødvendigt at tilsætte yderligere foder til beholderne under testen.
luminance of 350 ± 50 μE/m2/s, measured at the top of the canopy.
Fodring
The crop species are:
Der kan bruges foder, som er i stand til at opretholde populationen af enchytraeider.
tomato (Solanum lycopersicon);
Valsede havregryn, om muligt autoklaveret inden brug for at forebygge mikrobiel kontaminering (opvarmning er også hensigtsmæssig), har vist sig at være egnet foder.
cucumber (Cucumis sativus);
Foder gives først ved at blande 50 mg formalede valsede havregryn i jorden i hver beholder, inden ormene tilsættes.
lettuce (Lactuca sativa);
Derefter tilsættes der foder ugentligt frem til dag 21.
soybean (Glycine max);
Der tilsættes ikke foder på dag 28, da de voksne individer er blevet fjernet på dette trin, og deres afkom har brug for forholdsvis lidt supplerende foder efter dette tidspunkt.
cabbage (Brassica oleracea var. capitata);
Fodring under testen omfatter 25 mg formalede valsede havregryn pr. beholder, som forsigtigt anbringes på jordoverfladen for at undgå at skade ormene.
carrot (Daucus carota);
For at undgå svampevækst skal havreflagerne begraves i jorden ved at dække dem med små mængder jord.
oats (Avena sativa);
Hvis foder ikke bliver spist, skal rationen reduceres.
perennial ryegrass (Lolium perenne);
Design af testen til bestemmelse af dosisinterval
corn (Zea mays);
Når det er nødvendigt, udføres der en indledende test til bestemmelse af dosisinterval med f.eks. fem testkemikaliekoncentrationer på 0,1, 1,0, 10, 100 og 1000 mg/kg (tørvægt af jord).
onion (Allium cepa).
En replikat pr. behandling og kontrol er tilstrækkelig.
C.32.
Varigheden af den indledende test er to uger.
ENCHYTRAEID REPRODUCTION TEST
Ved afslutningen af testen vurderes dødeligheden for ormene.
This test method is equivalent to OECD test guideline (TG) 220 (2004).
En orm registreres som død, hvis den ikke reagerer på en mekanisk påvirkning af bagenden.
It is designed to be used for assessing the effects of chemicals on the reproductive output of the enchytraeid worm, Enchytraeus albidus Henle 1873, in soil.
Andre oplysninger end dødelighed kan også være afgørende for fastlæggelsen af koncentrationsområdet i den endelige test.
It is based principally on a method developed by the Umweltbundesamt, Germany (1) that has been ring-tested (2).
Ændringer i de voksne individers adfærd (f.eks. manglende evne til at grave sig ned i jorden; orme, der ligger ubevægelige op ad testbeholderens glasvæg) og morfologi (f.eks. åbne sår) registreres derfor også sammen med tilstedeværelsen af eventuelt afkom.
Other methods for testing the toxicity of chemicals to Enchytraeidae and earthworms have also been considered (3)(4)(5)(6)(7)(8).
Sidstnævnte kan bestemmes ved anvendelse af farvningsfremgangsmåden i tillæg 6.
Soil-dwelling annelids of the genus Enchytraeus are ecologically relevant species for ecotoxicological testing.
kan bestemmes omtrentligt ved at beregne det geometriske gennemsnit af dødelighedsdataene.
Whilst enchytraeids are often found in soils containing earthworms it is also true that they are often abundant in many soils where earthworms are absent.
Ved fastlæggelsen af koncentrationsområdet for den endelige test antages virkningerne på reproduktionen at være lavere end LC50 med højst en faktor 10.
Enchytraeids can be used in laboratory tests as well as in semi-field and field studies.
Der er dog tale om en empirisk sammenhæng, som ikke nødvendigvis er den samme i alle specifikke tilfælde.
From a practical point of view, many Enchytraeus species are easy to handle and breed, and their generation time is significantly shorter than that of earthworms.
Yderligere observationer foretaget i den indledende test, såsom forekomsten af afkom, kan bidrage til at præcisere det koncentrationsområde for testkemikaliet, der skal bruges til den endelige test.
The duration for a reproduction test with enchytraeids is therefore only 4-6 weeks while for earthworms (Eisenia fetida) it is 8 weeks.
Med henblik på nøjagtig bestemmelse af LC50 anbefales det at udføre testen med mindst fire replikater af hver af testkemikaliekoncentrationerne og et tilstrækkeligt antal koncentrationer til at give mindst fire statistisk signifikante forskellige gennemsnitsresponser ved disse koncentrationer.
Basic information on the ecology and ecotoxicology of enchytraeids in the terrestrial environment can be found in (9)(10)(11)(12).
Der bruges et tilsvarende antal koncentrationer og replikater til eventuelle kontroller.
Adult enchytraeid worms are exposed to a range of concentrations of the test chemical mixed into an artificial soil.
Design af den endelige formeringstest
The test can be divided into two steps:
Der foreslås tre design på grundlag af anbefalingerne som følge af en ringtest (2):
a range-finding test, in case no sufficient information is available, in which mortality is the main endpoint assessed after two weeks exposure and (b) a definitive reproduction test in which the total number of juveniles produced by parent animal and the survival of parent animals are assessed.
For at fastlægge NOEC skal der testes mindst fem koncentrationer i en geometrisk serie.
The duration of the definitive test is six weeks.
Der anbefales fire replikater for hver testkoncentration plus otte kontroller.
After the first three weeks, the adult worms are removed and morphological changes are recorded.
Koncentrationerne bør vælges med en afstandsfaktor, som ikke overstiger 1,8.
After an additional three weeks, the number of offspring, hatched from the cocoons produced by the adults, is counted.
For at bestemme ECx (f.eks. EC10, EC50) skal der testes mindst fem koncentrationer, og ECx skal ligge i det område, testkoncentrationerne dækker, således at ECx opnås ved interpolation og ikke ved ekstrapolation.
The reproductive output of the animals exposed to the test chemical is compared to that of the control(s) in order to determine (i) the no observed effect concentration (NOEC) and/or (ii) ECx (e.g. EC10, EC50) by using a regression model to estimate the concentration that would cause a x % reduction in reproductive output.
Der anbefales mindst fire replikater for hver testkoncentration og fire kontrolreplikater.
The test concentrations should bracket the ECx (e.g. EC10, EC50) so that the ECx then comes from interpolation rather than extrapolation.
Afstandsfaktoren kan variere, f.eks. under eller lig med 1,8 i det forventede effektområde og over 1,8 ved højere og lavere koncentrationer.
The water solubility, the log Kow, the soil water partition coefficient (e.g. Chapter C.18 or C.19 of this Annex) and the vapour pressure of the test chemical should preferably be known.
En kombineret metode giver mulighed for at bestemme både NOEC og ECx.
Additional information on the fate of the test chemical in soil, such as the rates of photolysis and hydrolysis is desirable.
Der anvendes otte behandlingskoncentrationer i en geometrisk serie.
This test method can be used for water soluble or insoluble chemicals.
Der anbefales fire replikater for hver behandling plus otte kontroller.
However, the mode of application of the test chemical will differ accordingly.
Der bruges ti voksne orme pr. testbeholder (se punkt 23).
The test method is not applicable to volatile chemicals, i.e. chemicals for which the Henry's constant or the air/water partition coefficient is greater than one, or chemicals for which the vapour pressure exceeds 0,0133 Pa at 25 °C.
Foder tilsættes ved begyndelsen af testen og herefter en gang om ugen (se punkt 29) frem til og med dag 21.
For the test to be valid, the following performance criteria should be met in the controls:
På dag 21 undersøges jordprøverne omhyggeligt med hænderne, levende voksne orme observeres og tælles, og ændringer i adfærd (f.eks. manglende evne til at grave sig ned i jorden; orme, der ligger ubevægelige op ad testbeholderens glasvæg) og morfologi (f.eks. åbne sår) registreres.
adult mortality should not exceed 20 % at the end of the range-finding test and after the first three weeks of the reproduction test.
Alle voksne orme fjernes derefter fra testbeholderne og testjorden.
assuming that 10 adults per vessel were used in setting up the test, an average of at least 25 juveniles per vessel should have been produced at the end of the test.
Testjord, der indeholder eventuelle kokoner, der måtte være frembragt, inkuberes i yderligere tre uger under samme testbetingelser, bortset fra at fodring kun finder sted på dag 35 (25 mg formalede valsede havregryn pr. beholder).
the coefficient of variation around the mean number of juveniles should not be higher than 50 % at the end of the reproduction test.
Efter seks uger tælles de nyudklækkede orme.
Where a test fails to meet the above validity criteria the test should be terminated unless a justification for proceeding with the test can be provided.
Denne metode, som er baseret på Rose Bengal-farvning (se tillæg 6), anbefales, men andre vådekstrations- og vådflotationsteknikker (uden varme) (se tillæg 6) har også vist sig at være egnede (4)(10)(11)(20).
The justification should be included in the test report.
Bengal Rose-farvning anbefales, fordi vådekstraktion fra et jordsubstrat kan hæmmes af turbiditet forårsaget af opslæmmede lerpartikler.
A reference chemical should be tested either at regular intervals or possibly included in each test to verify that the response of the test organisms has not changed significantly over time.
Hvis der ikke observeres virkninger ved den højeste koncentration i testen til bestemmelse af dosisinterval (dvs. 1000 mg/kg), kan formeringstesten udføres som en grænsetest med 1000 mg/kg, for at påvise at NOEC for formering er større end denne værdi.
A suitable reference chemical is carbendazim, which has been shown to affect survival and reproduction of enchytraeids (13)(14), or other chemicals whose toxicity data are well known could be also used.
Resume og tidsplan for testen
A formulation of carbendazim known by the trade name of Derosal™ supplied by AgrEvo Company (Frankfurt, Germany) and containing 360 g/l (32,18 %) active ingredient was used in a ring-test (2).
De enkelte trin i testen kan opsummeres som følger:
The EC50 for reproduction determined in the ring test was in the range of 1,2 ± 0,8 mg active ingredient (a.i) /kg dry mass (2).
Klokkeslæt
If a positive toxic standard is included in the test series, one concentration is used and the number of replicates should be the same as that in the controls.
Dag – 7 eller tidligere
For carbendazim, the testing of 1,2 mg a.i./kg dry weight (tested as a liquid formulation) is recommended.
Fremstil syntetisk jord (blanding af tørre bestanddele).
DESCRIPTION OF THE TEST
Dag – 5
Equipment
Kontroller den syntetiske jords pH
The test vessels should be made of glass or other chemically inert material.
Mål jordens maksimale vandholdende evne.
Glass jars (e.g. volume: 0,20 - 0,25 litre; diameter: ≈ 6 cm) are suitable.
Dag – 5 til – 3
The vessels should have transparent lids (e.g. glass or polyethylene) that are designed to reduce water evaporation whilst allowing gas exchange between the soil and the atmosphere.
Sorter orme med henblik på akklimatisering
The lids should be transparent to allow light transmission.
Dag – 3 til 0
Normal laboratory equipment is required, specifically the following:
Akklimatiser orme i mindst 24 timer
drying cabinet;
Dag – 1
stereomicroscope;
Fugt den syntetiske jord, og fordel den i partier
pH-meter and photometer;
Dag 0
suitable accurate balances;
Klargør stamopløsninger
adequate equipment for temperature control;
Tilsæt testkemikalie
adequate equipment for humidity control (not essential if exposure vessels have lids);
Afvej testsubstrat i testbeholdere
incubator or small room with air-conditioner;
Ibland foder
tweezers, hooks or loops;
Tilsæt orme
photo basin.
Mål jordens pH og fugtindhold
Preparation of the artificial soil
Dag 7
An artificial soil is used in this test (5)(7) with the following composition (based on dry weights, dried to a constant weight at 105 °C):
Kontroller jordens fugtindhold
10 % sphagnum peat, air-dried and finely ground (a particle size of 2 ± 1 mm is acceptable);
Fodr ormene
it is recommended to check that a soil prepared with a fresh batch of peat is suitable for culturing the worms before it is used in a test;
Dag 14
20 % kaolin clay (kaolinite content preferably above 30 %);
Bestem dødelighed for voksne individer
approximately 0,3 to 1,0 % calcium carbonate (CaCO3, pulverised, analytical grade) to obtain a pH of 6,0 ± 0,5;
Bestem antal unger
the amount of calcium carbonate to be added may depend principally on the quality/nature of the peat;
Dag 21
approximately 70 % air-dried quartz sand (depending on the amount of CaCO3 needed), predominantly fine sand with more than 50 % of the particles between 50 and 200 microns.
Observer de voksne individers adfærd
It is advisable to demonstrate the suitability of an artificial soil for culturing the worms and for achieving the test validity criteria before using the soil in a definitive test.
Fjern voksne individer
It is especially recommended to make such a check to ensure that the performance of the test is not compromised if the organic carbon content of the artificial soil is reduced, e.g. by lowering the peat content to 4-5 % and increasing the sand content accordingly.
Dag 28
By such a reduction in organic carbon content, the possibilities of adsorption of test chemical to the soil (organic carbon) may be decreased and the availability of the test chemical to the worms may increase.
Ingen fodring
It has been demonstrated that Enchytraeus albidus can comply with the validity criteria on reproduction when tested in field soils with lower organic carbon content than mentioned above (e.g. 2,7 %) (15), and there is experience — though limited — that this can also be achieved in artificial soil with 5 % peat.
Dag 35
When using natural soil in additional (e.g. higher tier) testing, the suitability of the soil and achieving the test validity criteria should also be demonstrated.
Dag 42
The dry constituents of the soil are mixed thoroughly (e.g. in a large-scale laboratory mixer).
Tæl unger
This should be done at least one week before starting the test.
Behandling af resultater
The mixed soil should be stored for two days in order to equilibrate/stabilise the acidity.
Tillæg 7 indeholder en oversigt, men testmetoden giver ingen endelig statistisk vejledning i analyse af testresultaterne.
For the determination of pH a mixture of soil and 1 M potassium chloride (KCl) or 0,01 M calcium chloride (CaCl2) solution in a 1:5 ratio is used (see (16) and Appendix 3).
I testen til bestemmelse af dosisinterval er det primære endepunkt dødelighed.
If the soil is more acidic than the required range (see paragraph 11), it can be adjusted by addition of an appropriate amount of CaCO3.
Ændringer i de voksne individers adfærd (f.eks. manglende evne til at grave sig ned i jorden; orme, der ligger ubevægelige op ad testbeholderens glasvæg) og morfologi (f.eks. åbne sår) registreres dog også sammen med tilstedeværelsen af eventuelle unger.
If the soil is too alkaline it can be adjusted by the addition of more of the mixture, referred to in paragraph 11, but excluding the CaCO3.
Der bruges normalt probabilistisk analyse (21) eller logistisk regression til at bestemme LC50.
The maximum water holding capacity (WHC) of the artificial soil is determined in accordance with procedures described in Appendix 2.
I tilfælde hvor denne analysemetode er uegnet (f.eks. hvis der er færre end tre koncentrationer med delvis dødelighed) kan der bruges alternative metoder.
One or two days before starting the test, the dry artificial soil is pre-moistened by adding enough de-ionised water to obtain approximately half of the final water content, that being 40 to 60 % of the maximum water holding capacity.
Disse metoder kan omfatte glidende gennemsnit (22), Trimmed Spearman-Karber-metoden (23) eller simpel interpolation (f.eks. geometrisk gennemsnit af LC0 og LC100 som beregnet ved kvadratroden af LC0 ganget med LC100).
At the start of the test, the pre-moistened soil is divided into portions corresponding with the number of test concentrations (and reference chemical where appropriate) and controls used for the test.
I den endelige test er endepunktet for testen frugtbarhed (dvs. antal unger).
The moisture content is adjusted to 40-60 % of the maximum WHC by the addition of the test chemical solution and/or by adding distilled or de-ionised water (see paragraphs 19-21).
Som i testen til bestemmelse af dosisinterval skal alle andre tegn på skade dog anføres i den endelige rapport.
The moisture content is determined at the beginning and at the end of the test (by drying to constant weight at 105 °C) and should be within the optimal range for the survival of the worms.
I forbindelse med den statistiske analyse er der brug for det aritmetiske gennemsnit og standardafvigelsen pr. behandling og pr. kontrol for at beregne reproduktionen.
A rough check of the soil moisture content can be obtained by gently squeezing the soil in the hand, if the moisture content is correct small drops of water should appear between the fingers.
Hvis der er foretaget en variansanalyse, kan standardafvigelsen, s, og frihedsgraderne, df, erstattes af henholdsvis det samlede variansestimat fra variansanalysen og frihedsgraderne herfra — forudsat at variansen ikke afhænger af koncentrationen.
Selection and preparation of test animals
I dette tilfælde bruges kontrollernes og behandlingernes individuelle varianser.
The recommended test species is Enchytraeus albidus Henle 1837 (white potworm), a member of the family Enchytraeidae (order Oligochaeta, phylum Annelida).
Disse værdier beregnes normalt med kommerciel statistisk software, der bruger resultater pr. beholder som replikater.
E. albidus is one of the largest species of enchytraeids, with specimens of up to 35 mm in length being recorded (17)(18).
Hvis det skønnes mere hensigtsmæssigt at samle data for de negative kontroller og opløsningsmiddelkontrollerne end at teste dem hver for sig, skal de testes, for at konstatere at de ikke er signifikant forskellige (punkt 45 og tillæg 7 indeholder beskrivelser af egnede test).
E. albidus has a world-wide distribution and is found in marine, freshwater and terrestrial habitats, mainly in decaying organic matter (seaweed, compost) and rarely in meadows (9).
Beregning af NOEC
E. albidus is commercially available, as a fish food.
Anvendelse af stærke statistiske test foretrækkes.
It should be checked whether the culture is contaminated by other, usually smaller, species (1) (19).
Der bør bruges oplysninger fra f.eks. tidligere erfaring med ringtest eller andre historiske oplysninger om, hvorvidt data er nogenlunde normalt fordelt.
If contamination occurs, all worms should be washed with water in a petri dish.
Varianshomogenitet (homoskedasitet) er mere afgørende.
Large adult specimens of E. albidus should then be selected (using a stereomicroscope) to start a new culture and all other worms are discarded.
Erfaringer viser, at variansen ofte stiger med stigende gennemsnit.
E. albidus can be bred easily in a wide range of organic materials (see Appendix 4).
I disse tilfælde kan transformation af data medføre homoskedasitet.
The life-cycle of E. albidus is short since maturity is reached between 33 days (at 18 °C) and 74 days (at 12 °C) (1).
En sådan transformation bør dog være baseret på erfaring med historiske data snarere end på data, der undersøges.
Only cultures that have been kept without problems in the laboratory for at least 5 weeks (one generation) will be used for the test.
Med homogene data udføres flere t-test, såsom Williams' test (α = 0,05, ensidet) (24)(25) eller i visse tilfælde Dunnetts test (26)(27).
Other species of the Enchytraeus genus are also suitable, e.g. E. buchholzi Vejdovsky 1879 or E. crypticus Westheide & Graefe 1992 (see Appendix 5).
Det skal bemærkes, at t-værdierne i tabellen — i tilfælde af ulige replikation — skal korrigeres i henhold til Dunnett og Williams.
If other species of Enchytraeus are used, they must be clearly identified and the rationale for the selection of the species should be reported.
Nogle gange stiger/falder responserne ikke regelmæssigt på grund af stor variation.
The animals used in the tests are adult worms.
I dette tilfælde, hvor der er tale om en kraftig afvigelse fra monotoni, er Dunnetts test mere velegnet.
They should have eggs (white spots) in the clitellum region, and they should be approximately the same size (about 1 cm long).
Hvis der er afvigelser fra homoskedasitet, kan det være fornuftigt at undersøge mulige virkninger på varians nærmere, for at beslutte om der kan bruges t-test uden at miste betydelig styrke (28).
Synchronisation of the breeding culture is not necessary.
Er der udført en grænsetest, og forudsætningerne for parametriske testprocedurer (normalitet, homogenitet) er opfyldt, kan den parvise Student-t-test bruges eller alternativt Mann-Whitney-U-testen (29).
If the enchytraeids are not bred in the same soil type and under the conditions (including feeding) used for the final test they must be acclimatised for at least 24 hours and up to three days.
Beregning af ECx
A larger number of adults than that needed for performing the test should initially be acclimatised to allow scope for rejection of damaged or otherwise unsuitable specimens.
For at beregne enhver ECx-værdi bruges middelværdierne pr. behandling til regressionsanalyse (lineær eller ikke-lineær), når der er opnået et passende dosis/respons-forhold.
At the end of the acclimatisation period, only worms containing eggs and exhibiting no behavioural abnormalities (e.g. trying to escape from the soil) are selected for the test.
For ormenes vækst som en kontinuerlig respons kan ECx-værdier estimeres ved hjælp af passende regressionsanalyse (35).
The worms are carefully removed using jeweller's tweezers, hooks or loops and placed in a petri dish containing a small amount of fresh water.
Blandt egnede forhold for kvantale data (dødelighed/overlevelse) og afkom er normale sigmoidforhold, logistiske forhold eller Weibull-forhold, som omfatter to til fire parametre, hvoraf nogle også kan modellere hormetiske responser.
Reconstituted fresh water as proposed in Chapter C.20 of this Annex (Daphnia magna Reproduction Test) is preferred for this purpose since de-ionised, de-mineralised or tap water could be harmful to the worms.