natgillin/translations-raw huggingface dataset:
Fisken vejes i våd tilstand.
A reduction in the circulating level of oestrogen in females, for instance through the inhibition of the aromatase converting the endogenous androgen to the natural oestrogen 17β-oestradiol, causes a decrease in the VTG concentration, which is used to detect chemicals having aromatase inhibiting properties or steroidogenesis inhibitors more broadly (33).
Med henblik på fremstilling af væv til VTG-analyse kan fisken anbringes på en korkplade på præparatbordet i et dissektionsmikroskop.
The biological relevance of the VTG response following oestrogenic/aromatase inhibition is established and has been broadly documented (8) (9).
a. For zebrafisk skæres hovedet lige bag brystfinnen, og halen skæres lige bag rygfinnen.
However, it is possible that production of VTG in females can also be affected by general toxicity and non-endocrine toxic modes of action.
b. For japansk risfisk åbnes abdomen ved en omhyggelig incision, der strækker sig langs den ventrale midterlinje fra skulderåget til et punkt lige oven for gattet.
Several measurement methods have been successfully developed and standardised for routine use to quantify VTG in blood, liver, whole body or head/tail homogenate samples collected from individual fish.
Ved hjælp af små pincetter og en lille saks fjernes leveren forsigtigt.
This is the case for zebrafish, three-spined stickleback and Japanese medaka and also the partially validated species fathead minnow;
Prøver til VTG-analyse anbringes i eppendorfrør og fryses straks i flydende kvælstof.
species-specific Enzyme-Linked Immunosorbent Assay (ELISA) methods using immunochemistry for the quantification of VTG are available (5) (10) (11) (12) (13) (14) (15) (16).
Kadaveret, herunder kønskirtlerne, anbringes i en på forhånd mærket vævskassette af plast, som overføres til Davidson's eller Bouin's fikseringsmiddel.
In Japanese medaka and zebrafish, there is a good correlation between VTG measured from blood plasma, liver and homogenate samples although homogenates tend to show slightly lower values than plasma (17) (18) (19).
Fikseringsmidlets volumen skal være mindst 10 gange den tilnærmede vævsvolumen.
Appendix 5 provides the recommended procedures for sample collection for VTG analysis.
Fikseringsbeholderen rystes forsigtigt i fem sekunder for at fjerne luftbobler fra kassetten.
Change in the phenotypic sex ratio (proportions of sex) is an endpoint reflecting sex reversal.
a. Alt væv bliver liggende i Davidson's fikseringsmiddel natten over, hvorefter det den næste dag overføres til individuelle beholdere med 10 % neutralbufret formalin.
In principle, oestrogens, anti-oestrogens, androgens, anti-androgens and steroidogenesis inhibiting chemicals can affect the sex ratio of developing fish (20).
Kassettebeholderne rystes forsigtigt i fem sekunder for at sikre en tilstrækkelig gennemtrængning af formalin i kassetterne.
It has been shown that this sex reversal is partly reversible in zebrafish (21) following oestrogen-like chemical exposure, whereas sex reversal following androgen-like chemical exposure is permanent (30).
b. Vævene bliver liggende i Bouins fikseringsmiddel i 24 timer, hvorefter de overføres til 70 % ethanol.
The sex is defined as female, male, intersex (both oocytes and spermatogenetic cells in one gonad) or undifferentiated, determined in individual fish via histological examination of the gonads.
Vævsbehandling
Guidance is given in Appendix 7 and in the OECD Guidance Document on the Diagnosis of Endocrine-Related Histopathology of Fish Gonads (22).
at tørre væv med henblik på tilstrækkelig gennemtrængning af paraffin
Genetic sex is examined via genetic markers when they exist in a given fish species.
at behandle vævet med paraffin for at opretholde vævets integritet og skabe en fast overflade til mikrotomi.
In Japanese medaka the female XX or male XY genes can be detected by Polymerase Chain-Reaction (PCR), or the Y-linked DM domain gene (DMY) can be analysed (DMY negative or positive) as described in (23) (24).
Mærkede vævskassetter fjernes fra opbevaringen i formalin/ethanol, og kassetterne anbringes i behandlingskurven/-ene.
In three-spined stickleback, there is an equivalent PCR method for genetic sex determination described in Appendix 10.
Behandlingskurven indsættes i vævsbehandlingsenheden.
Where the genetic sex can be individually linked to the phenotypic sex, the power of the test is improved and therefore genetic sex should be determined in species with documented genetic sex markers.
Behandlingsplanen vælges.
The two core endocrine endpoints, VTG and sex ratio, can in combination demonstrate the endocrine mode of action (MOA) of the chemical (Table 1).
Når vævsbehandlingsenheden har afsluttet behandlingscyklussen, kan kurven/-ene overføres til den indlejrede station.
The sex ratio is a population relevant biomarker (25) (26) and for some well defined modes of action, the FSDT results may be used for hazard and risk assessment purposes when deemed appropriate by the regulatory agency.
Indlejring
These modes of action are at present oestrogens, androgens and steroidogenesis inhibitors.
Formål:
Reaction of the endocrine endpoints to different modes of action of chemicals
at vende prøven korrekt i størknet paraffin med henblik på mikrotomi.
↑ = increasing, ↓ = decreasing, — = not investigated
Kassettekurven/-ene fjernes fra behandlingsenheden og nedsænkes i det paraffinfyldte forkammer i indlejringsstationens varmekonsol, eller kassetterne flyttes til en separat paraffinvarmer.
References
Den første kassette, der skal indlejres, fjernes fra forkammeret i varmekonsollen eller paraffinvarmeren.
Weak oestrogen agonist
Kassettelåget fjernes og kasseres, og kassettemærket kontrolleres i forhold til dyrejournalerne for at løse potentielle uoverensstemmelser før indlejring.
↑♀ or ↑Undiff
Der vælges en indlejringsform i passende størrelse.
Strong oestrogen agonist
Formen holdes under dispenserens tud og fyldes med smeltet paraffin.
↑♀ or ↑Undiff, No ♂
Prøven fjernes fra kassetten og anbringes i den smeltede paraffin i formen.
Oestrogen antagonist
Dette gentages med 4-8 prøver for hver paraffinform.
↓♀, ↑Undiff.
Den enkelte fisks placering afmærkes ved at anbringe fisk nr. 1 i en vinkel på 180 grader i forhold til fisk nr. 2-4/8.
Androgen agonist
Ekstra paraffin tilsættes for at dække prøven.
↑♂, No ♀
Formen med kassettebasen anbringes på kryokonsollens køleplade.
Androgen antagonist
Når paraffinen er størknet, fjernes blokken (dvs. den hærdede paraffin indeholdende væv og kassettebase) fra formen.
↑Intersex
Mikrotomi
Aromatase inhibitor
Skær og anbring histologiske snit til farvning.
The FSDT does not cover the reproductive life stage of the fish and therefore chemicals that are suspected to affect reproduction at lower concentrations than sexual development should be examined in a test that covers reproduction.
Den første fase af mikrotomien, der kaldes »facing«, udføres som følger:
Definitions for the purpose of this Test Method are given in Appendix 1.
a. Paraffinblokken anbringes i mikrotomens kæber.
The in vivo FSDT is intended to detect chemicals with androgenic and oestrogenic properties as well as anti-androgenic, anti-oestrogenic and steroidogenesis inhibiting properties.
b. Kæberne føres frem ved at rotere mikrotomhjulet, og der skæres tykke snit af blokkens paraffinoverflade, indtil kniven når de indlejrede væv.
The FSDT validation phases (1 and 2) did cover oestrogenic, androgenic and steroidogenesis inhibiting chemicals.
c. Snittykkelsen på mikrotomen sættes til 3-5 mikron.
The effects in the FSDT of oestrogen- and androgen antagonists can be seen in Table 1 but these MOA are less documented at present time.
Kæberne føres frem, og der skæres flere snit af blokken for at fjerne eventuelle artefakter, der har dannet sig på vævets snitflade under grovtrimming.
In the test, fish are exposed, from newly fertilised egg until the completion of sexual differentiation, to at least three concentrations of the test chemical dissolved in water.
d. Blokken kan fjernes fra kæberne og anbringes med oversiden nedad på isen for at gennemvæde vævet.
The test conditions should be flow-through unless not possible due to the availability or nature (e.g. limited solubility) of the test chemical.
Den næste fase af mikrotomien er skæring af endeligt snit og montering af vævssnit på objektglas.
The test starts with the placing of newly fertilised eggs (before cleavage of the blastodisc) in the test chambers.
Disse procedurer udføres som følger:
The loading of the chambers is described for each species in paragraph 27.
a. Hvis blokken er blevet sat på is, fjernes blokken fra isen og anbringes igen på mikrotomens kæber.
For the validated fish species, Japanese medaka, the three-spined stickleback and zebrafish, the test is terminated at 60 dph.
b. Mikrotomens snittykkelse sættes til 3-5 mikron, og kæberne føres frem ved at dreje mikrotomhjulet.
At test termination, all fish are euthanised humanely.
Der skæres snit af blokken, indtil der er frembragt et »bånd« med mindst et acceptabelt snit, der indeholder kønskirtler.
A biological sample (blood plasma, liver or head/tail homogenate) is collected for VTG analysis from each fish and the remaining part is fixed for histological evaluation of the gonads to determine the phenotypic sex;
(Under delingen kan blokken efter behov fjernes fra kæberne, anbringes på isen for at gennemvæde vævet og sættes tilbage i kæberne igen.)
optionally, histopathology (e.g. staging of gonads, severity of intersex) can be performed.
c. Snittene anbringes fladt flydende på vandoverfladen i vandbadet.
A biological sample (the anal- or the dorsal fin) for the determination of the genetic sex is taken in species possessing appropriate markers (Appendices 9 and 10).
Der gøres forsøg på at få mindst et snit, som ikke indeholder folder, og hvor der ikke er opsamlet luftbobler under det.
An overview of relevant test conditions specific for validated species:
d. Et mikroskopobjektglas nedsænkes under det bedste snit, som løftes op af vandet ved hjælp af glasset.
Japanese medaka, the three-spined stickleback and zebrafish is provided in Appendix 2.
Denne proces kaldes »montering« af snittet på glasset.
Results from an acute toxicity test or other short-term toxicity assay [e.g. test method C.14 (34) and OECD TG 210 (1)], preferably performed with the species chosen for this test, should be available.
e. Der klargøres tre snit til et sæt fisk.
This implies that the water solubility and the vapour pressure of the test chemical are known and a reliable analytical method for the quantification of the chemical in the test chambers, with known and reported accuracy and limit of detection, is available.
Det andet og tredje snit udtages med intervaller på 50 mikron efter det første snit.
Other useful information includes the structural formula, purity of the chemical, stability in water and light, pKa, Pow and results of a test for ready biodegradability (Test Method C.4) (35).
Hvis fiskene ikke er indlejret med deres kønskirtler i det samme snitniveau, skal der foretages flere snit for at sikre, at der fås mindst seks snit, der indeholder kønskirtler, fra hver fisk.
Test acceptance criteria
f. Med en opmærkningspen til objektglas registreres det bloknummer, glasset blev fremstillet med, på glasset.
The dissolved oxygen concentration should be at least 60 per cent of the air saturation value (ASV) throughout the test;
g. Glasset anbringes i et farvningsstativ.
The water temperature should not differ by more than ± 1,5 °C between test chambers at any one time during the exposure period and be maintained within the temperature ranges specified for the test species (Appendix 2);
h. Blokken fjernes fra kæberne og anbringes med oversiden nedad med henblik på opbevaring.
A validated method for analysis of the exposure chemical with a detection limit well below the lowest nominal concentration should be available and evidence should be gathered to demonstrate that the concentrations of the test chemical in solution have been satisfactorily maintained within ± 20 % of the mean measured values;
Farvning, anvendelse af dækglas og glasmærkning
Overall survival of fertilised eggs in the controls and, where relevant, in the solvent controls, should be greater than or equal to the limits defined in Appendix 2;
at farve snittene til histopatologisk undersøgelse
Acceptance criteria related to growth and proportions of sex at termination of the test are based on data from the control groups (pooled solvent and water control unless they are significantly different, then solvent only):
at forsegle monterede og farvede væv permanent
Japanese medaka
at identificere farvede snit permanent på en måde, der sikrer fuld sporbarhed.
Three-spined stickleback
Farvning
Growth
a. Objektglas lufttørres natten over før farvning.
Fish wet weight, blotted dry
b. Snittene farves med hematoxylin-eosin.
> 75 mg
Anvendelse af dækglas
Length (standard length)
a. Dækglas kan anvendes manuelt eller automatisk.
> 20 mm
b. Et objektglas dyppes i xylen eller TissueClear, og overskydende xylen/TissueClear bankes forsigtigt af glasset.
> 14 mm
c. Ca. 0,1 ml monteringsmedium påføres nær enden af objektglasset modsat den matte ende eller på dækglasset.
Sex ratio (% males or females)
d. Dækglasset hældes i en lav vinkel, når det lægges på objektglasset.
When a solvent is used it should have no statistical significant effect on survival and should not produce any endocrine disrupting effects or other adverse effects on the early-life stages as revealed by a solvent control.
Mærkning
If a deviation from the test acceptance criteria is observed, the consequences should be considered in relation to the reliability of the test data and these considerations should be included in the reporting.
a. Hvert objektglasmærke bør indeholde følgende oplysninger.
Test chambers
i. Laboratoriets navn
Any glass, stainless steel or other chemically inert chambers can be used.
Prøvenr./objektglasnr.
The dimensions of the chambers should be large enough to allow compliance with loading rate criteria given below.
Kemikalie/behandlingsgruppe
It is desirable that test chambers be randomly positioned in the test area.
Statistisk flowdiagram for vitellogeninanalyse
A randomised block design with each concentration being present in each block is preferable to a completely randomised design.
>= 4 replikater pr. konc.
The test chambers should be shielded from unwanted disturbance.
Replikatgennemsnit normalt fordelt
Recommended fish species are given in Appendix 2.
Replikatgennemsnit ikke normale eller ikke homogene
The procedures for inclusion of new species are given in paragraph 2.
Dunnetts test på indlejret ANOVA
Holding of parental fish
Dunnetts test
Details on holding the parental fish under satisfactory conditions may be found in OECD TG 210(1).
<= 3 replikater pr. konc.
Parental fish should be fed once or twice a day with appropriate food.
Dunns test
Handling of embryos and larvae
Undlad vandkontrol
Initially, embryos and larvae may be exposed within a main chamber in smaller glass or stainless steel chambers, fitted with mesh sides or ends to permit a flow of test chemical through the chamber.
Dunns test på repli-katgennemsnit Dunns test
Non-turbulent flow through these small chambers may be induced by suspending them from an arm arranged to move the chamber up and down but always keeping the organisms submerged.
Step-down Jonckheere-test eller Williams' test
Where egg containers, grids or meshes have been used to hold eggs within the main test chamber, these restraints should be removed after the larvae hatch, except that meshes should be retained to prevent the escape of the fish.
Kombiner kontroller, bevar undergrupper
If there is a need to transfer the larvae, they should not be exposed to the air and nets should not be used to release fish from egg containers.
Sammenlign kontroller ved hjælp af Wilcoxon- eller t-test.
The timing of this transfer varies with the species and transfer may not always be necessary.
Er kontrollerne forskellige?
Any water in which the test species shows control survival at least as good as in water described in Appendix 3 is suitable as test water.
Statistisk flowdiagram for analyse af kønsfordeling
In order to ensure that the dilution water will not unduly influence the test result (for example by reacting with the test chemical) or adversely affect the performance of the brood stock, samples should be taken at intervals for analysis.
Anvend step-down Jonckheere- Terpstra-test (+) til at bestemme NOEC
Total organic carbon, conductivity, pH and suspended solids should be measured, for example every three months where dilution water is known to be relatively constant in quality.
Brug Dunns test eller Mann-Whitney-U-test med Bonferroni-Holm-tilpasning til at bestemme NOEC
Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (e.g. Ca2+, Mg2+, Na+, K+, Cl–, SO42–) and pesticides should be done, if water quality is questionable.
Brug Dunnetts test, hvis varianserne er homogene (*), og ellers Tamhane-Dunnett (T3)-test til at bestemme NOEC
Details about chemical analysis and water collection can be found in paragraph 34.
Er data normalt fordelt?
Flow-through system should be used if practically possible.
Stemmer data overens med monotont dosis/respons-forhold?
For flow-through tests, a system that continually dispenses and dilutes a stock solution of the test chemical (e.g. metering pump, proportional diluter, and saturator system) is necessary to deliver a series of concentrations to the test chambers.
(‡) Eller anden anerkendt kontroludvælgelse
The flow rates of stock solutions and dilution water should be checked at intervals during the test and should not vary by more than 10 % throughout the test.
Efter arcsin-sqrt-transformation
A flow rate equivalent to at least five test chamber volumes per 24 hours has been found suitable (1).
(+) Hvis der er under fem forsøgsenheder pr. behandling, bør der anvendes eksakt J-T- eller M-W-test, hvis tilgængelig
Care should be taken to avoid the use of plastic tubing or other materials, some of which may contain biologically active chemicals or may adsorb the test chemical.
Kombiner kontroller
The stock solution should preferably be prepared without the use of solvents by simply mixing or agitating the test chemical in the dilution water by using mechanical means (e.g. stirring or ultrasonication).
Undlad vandkontrol (‡)
If the test chemical is difficult to dissolve in water, procedures described in the OECD Guidance Document on aquatic toxicity testing of difficult substances and mixtures should be followed (36).
Sammenlign kontroller ved hjælp af t-test.
The use of solvents should be avoided but may be necessary in some cases in order to produce a suitably concentrated stock solution.
Anvendes der opløsningsmiddel?
Examples of suitable solvents are given in (36).
Tillæg 9
Semi-static test conditions should be avoided unless justification is provided on compelling reasons associated with the test chemical (e.g. stability, limited availability, high cost or hazard).
Vejledning i udtagning af vævsprøver til genetisk kønsbestemmelse og til genetisk kønsbestemmelse efter PCR-metoden
For the semi-static technique, two different renewal procedures may be followed.
Udtagning af vævsprøver, klargøring og opbevaring før bestemmelse af genetisk køn efter PCR-metoden hos risfisk (fremstillet af Laboratory for Aquatic Organisms of Bayer CropScience AG)
Either new test solutions are prepared in clean chambers and surviving eggs and larvae gently transferred into the new chambers, or the test organisms are retained in the test chambers whilst a proportion (at least two thirds) of the test water is changed daily.
Med en fin saks klippes gat- eller rygfinne af hver fisk og anbringes i et rør fyldt med 100 μl ekstraktionsbuffer 1 (se oplysninger om fremstilling af buffer nedenfor).
Conditions of Exposure
Saksen rengøres efter hver fisk i et bæger fyldt med destilleret H2O og tørres med en papirserviet.
Collection of eggs and duration
Nu homogeniseres finnevævet i et mikrorør med teflonpistil med henblik på lysis af celler.
To avoid genetic bias, eggs are collected from a minimum of three breeding pairs or groups, mixed and randomly selected to initiate the test.
Til hvert rør anvendes en ny pistil for at forebygge eventuel kontaminering.
For the three-spined stickleback, see the description of artificial fertilisation in Appendix 11.
Pistillerne anbringes natten over i 0,5 M NaOH, skylles i fem minutter i destilleret H2O og opbevares indtil brug i ethanol eller sterilt efter autoklavering.
The test should start as soon as possible after the eggs have been fertilised, the embryos preferably being immersed in the test solutions before cleavage of the blastodisc commences, or as close as possible after this stage and no later than 12 h post fertilisation.
Det er også muligt at opbevare finnevævet uden ekstraktionsbuffer 1 på tøris og siden i fryseskab ved – 80 °C for at forhindre degeneration af DNA.
The test should continue until sexual differentiation in the control group is completed (60 dph for Japanese medaka, the three-spined stickleback and zebrafish).
Men ekstraktionen forløber bedre, hvis man ekstraherer DNA samtidig (se ovenfor vedrørende håndtering.
Loading
Prøver optøs på is efter opbevaring ved – 80 °C, før bufferen fyldes i rørene).
The number of fertilised eggs at the start of the test should be at least 120 per concentration divided between a minimum of 4 replicates (square root allocation to control is accepted).
Efter homogenisering lægges alle rør i et vandbad og koges i 15 minutter ved 100 °C.
The eggs should be randomly distributed (by using statistical tables for randomisation) among treatments.
Herefter pipetteres 100 μ ekstraktionsbuffer 2 (se nærmere oplysninger om fremstilling af buffer nedenfor) i hvert rør.
The loading rate (for definition, see Appendix 1) should be low enough in order that a dissolved oxygen concentration of at least 60 % of the ASV can be maintained without direct aeration of the chambers.
Prøverne opbevares ved stuetemperatur i 15 minutter, og i mellemtiden rystes de undertiden forsigtigt i hånden.
For flow-through tests, a loading rate not exceeding 0,5 g/l per 24 hours, and not exceeding 5 g/l of solution at any time is recommended.
Efterfølgende lægges alle rør i vandbadet igen og koges i endnu 15 minutter ved 100 °C.
No later than 28 days post fertilisation the number of fish per replicate should be redistributed, so that each replicate contains as equal a number of fish as possible.
Indtil videre analyse fryses rørene ved – 20 °C.
If exposure related mortality occurs, the number of replicates should be reduced appropriately so that fish density between treatment levels is kept as equal as possible.
Fremstilling af buffer:
The photoperiod and water temperature should be appropriate for the test species (see Appendix 2 for experimental conditions for the FSDT).
500 mg N-Lauroylsarcosin (f.eks. Merck KGaA, Darmstadt, GE)
Food and feeding are critical, and it is essential that the correct food for each stage is supplied at appropriate time intervals and at a level sufficient to support normal growth.
→ autoklaveres
Feeding should be ad libitum whilst minimising the surplus.
20 g Chelex (f.eks. Biorad, München, GE)
To obtain a sufficient growth rate, fish should be fed at least twice daily (accepting once daily on weekends), separated by at least three hours between each feed.
skal kvælde i 100 ml dest.
Surplus food and faeces should be removed, as necessary, to avoid accumulation of waste.
Til PCR bruges den klare supernatant separeret fra bundfaldet.
As experience is gained, food and feeding regimes are continuously being refined to improve survival and optimise growth.
Det skal absolut undgås, at eventuelle spor af Chelex (lokaliseret i bundfaldet) overføres til PCR-reaktionen, fordi dette vil forstyrre »Taq«-polymeraseaktiviteten.
Effort should therefore be made to confirm the proposed regime with acknowledged experts.
Supernatanten bruges direkte eller kan opbevares frosset (ved – 20 °C) og genoptøs til senere analyser over flere cyklusser uden negativ indvirkning på DNA.
Feeding should be withheld 24 hours before ending the test.