natgillin/translations-raw huggingface dataset:
Fremstilling af »reaktionsblandingen« (25 μl pr. prøve):
Examples of appropriate food items are listed in Appendix 2 (see also the OECD Fish Testing Framework (39).
Mængde
Test chemicals should be spaced as described in Appendix 4.
Slutkoncentration
A minimum of three test concentrations in at least four replicates should be used.
DNA-template
The curve relating LC50 to period of exposure in the acute studies available should be considered when selecting the range of test concentrations.
0,5 μl-2 μl
Five test concentrations are recommended if the data are to be used for risk assessment.
10 x PCR-buffer med MgCl2
Concentrations of the chemical higher than 10 % of the acute adult LC50 or 10 mg/l, whichever is the lower, need not be tested.
2,5 μl
The maximum test concentration should be 10 % of the LC50 on the larval/juvenile life-stage.
Nukleotider (hver af dATP, dCTP, dGTP, dTTP)
Controls
Fremadrettet primer (10μM) (se nedenfor 3-5)
A dilution water control (≥ 4 replicates) and, if relevant, a solvent control (≥ 4 replicates) should be run in addition to the test concentrations.
0,5 μl
Only solvents that have been investigated not to have any statistical significant influence on the test endpoints should be used in the test.
Bagudrettet primer (10 μM) (se nedenfor 3-5)
Where a solvent is used, its final concentration should not be greater than 0,1 ml/l (36) and it should be the same concentration in all test chambers, except the dilution water control.
Vand (PCR-kvalitet)
However, every effort should be made to avoid the use of such solvent or keep solvent's concentrations to a minimum.
op til 25 μl
Frequency of Analytical Determinations and Measurements
0,3 μl
Chemical analysis of the test chemical concentration should be performed before initiation of the test to check compliance with the acceptance criteria.
10 x PCR-buffer med MgCl2:
All replicates should be analysed individually at the beginning and termination of the test.
670 mM Tris/HCl (pH 8,8 ved 25 °C), 160 mM (NH4)2SO4, 25 mM MgCl2, 0,1 %Tween 20.
One replicate per test concentration should be analysed at least once per week during the test, changing systematically between replicates (1,2,3,4,1,2…).
Til hver PCR (se nedenfor 3-5) er der brug for den særlige primer som en ny kombination af »reaktionsblanding« og den tilstrækkelige nødvendige mængde DNA-template til hver prøve (se ovenfor).
If samples are stored to be analysed at a later time, the storage method of the samples should be previously validated.
De respektive mængder overføres til nye rør ved hjælp af pipetter.
Samples should be filtered (e.g. using a 0,45 μm pore size) or centrifuged to ensure that the determinations are made on the chemical in true solution.
Herefter lukkes alle rør, omrøres (ca. 10 sek.) og centrifugeres (10 sek. ved stuetemperatur).
During the test, dissolved oxygen, pH, total hardness, conductivity, salinity (if relevant), and temperature should be measured in all test chambers.
Nu kan de respektive PCR-programmer startes.
As a minimum dissolved oxygen, salinity (if relevant), and temperature should be measured weekly, and pH, conductivity and hardness at the beginning and at the end of the test.
Der benyttes yderligere en positiv kontrol (eksemplarisk DNA-prøve med kendt aktivitet og klare resultater) og en negativ kontrol (1 μl dest.
Temperature should preferably be monitored continuously in at least one test chamber.
H2O) i hvert PCR-program.
Results should be based on measured concentrations.
Fremstilling af agarosegel (1 %) — Under igangværende PCR-programmer:
However, if the concentration of the test chemical in solution has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test, then the results can either be based on nominal or measured values.
Opløs 3 g agarose i 300 ml 1 × TAE-buffer (1 % agarosegel).
Observations and measurements
Denne opløsning koges i en mikrobølgeovn (ca. 2-3 min.).
Stage of embryonic development
Overfør den varme opløsning til en særlig støbningsboks, der ligger på is.
The exposure should begin as soon as possible after fertilisation and before cleavage of the blastodisc commences and no later than 12 h post fertilisation to ensure exposure during early embryonic development.
Efter ca. 20 min. er agarosegelen klar til brug.
Hatching and survival
»Mact1(øvre/fremadrettet)« → TTC AAC AGC CCT GCC ATG TA
for eggs:
»Mact2(nedre/bagudrettet)« → GCA GCT CAT AGC TCT TCT CCA GGG AG
particularly in the early stages, a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance;
Program:
for larvae and juvenile fish:
Cyklus (35-gange):
immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque coloration of central nervous system and/or lack of reaction to mechanical stimulus.
→ 45 sek. ved 56 °C
It should be noted that abnormal embryos and larvae occur naturally and can be of the order of several per cent in the control(s) in some species.
»PG 17,6« (nedre/bagudrettet) → GAT CGT CCC TCC ACA GAG AAG AGA
These observations will result in some or all of the following data being available for reporting:
Cyklus (40-gange):
cumulative mortality;
→ 45 sek. ved 55 °C
numbers of healthy fish at end of test;
→ 1 min. 30 sek. ved 68 °C
time to start of hatching and end of hatching;
Y-gen-PCR-program som »kontrol« for X- og Y-gen-PCR-programmet:
length and weight of surviving animals;
»DMTYa (øvre/fremadrettet)« → GGC CGG GTC CCC GGG TG
numbers of deformed larvae;
»DMTYd (nedre/bagudrettet)« → TTT GGG TGA ACT CAC ATG G
numbers of fish exhibiting abnormal behaviour.
Farvning af PCR-prøver:
Sampling of fish
Farvningsopløsning:
Fish sampling is performed at termination of the test.
0,25 % bromphenolblå
Sampled fish should be euthanised with e.g. MS-222 (100-500 mg per l buffered with 200 mg NaHCO3 per l) or FA-100 (4-allyl-2-methoxyphenol: eugenol) and individually measured and weighed as wet weight (blotted dry) or anaesthetised if a blood sample should be taken (see paragraph 49).
0,25 % xylencyanol
Sampling for VTG analysis and sex determination via histological evaluation
Tilsæt 1 μl farvningsopløsning i hvert enkelt rør ved hjælp af en pipette
All fish should be sampled and prepared for analysis of sex and VTG.
Påbegyndelse af gelelektroforesen:
All fish should be analysed histologically to determine sex.
Den fremstillede 1 % agarosegel overføres i et gelelektroforesekammer fyldt med 1 × TAE-buffer
For the VTG measurements, a sub-sampling of at least 16 fish from each replicate is accepted.
10-15 μl af hver farvet PCR-prøve pipetteres i en fordybning i agarosegelen.
More fish should be analysed for VTG if the results of the sub-sampling turn out to be unclear.
Der pipetteres også 5-15 μl 1kb-»Ladder« (Invitrogen) i en separat fordybning
The sampling procedure for VTG and sex determination is dependent on the VTG analysis method:
Start elektroforesen ved 200 V
Head/tail homogenate method for VTG analysis
Stop efter 30-45 min.
The fish is euthanised.
Bestemmelse af båndene:
Head and tail of each fish are separated from the body of the fish by cuts made right behind the pectoral fins, and right behind the dorsal fin, using a scalpel (See Figure 1).
Rens agarosegelen i destilleret H2O.
The head and tail part from each fish are pooled, weighed and individually numbered, frozen in liquid nitrogen and stored at – 70° or less for VTG analysis.
Overfør nu agarosegelen i ethidiumbromid i 15-30 min.
The body part of the fish is numbered and fixed in an appropriate fixative for histological evaluation (22).
Tag herefter et billede af agarosegelen i en UV-lysboks.
By use of this method VTG and histopathology are evaluated on each individual and a possible change in the VTG level can thus be related to the phenotypic sex of the fish or genetic sex (Japanese medaka and the three-spined stickleback) of the fish.
Endelig analyseres prøverne sammenlignet med det/de positive kontrolbånd og ladder.
For further information see guidance for homogenisation (Appendix 5) and guidance for VTG quantification (Appendix 6).
Tillæg 10
Liver homogenate method for VTG analysis
Vejledning i udtagning af vævsprøver til genetisk kønsbestemmelse efter PCR-metoden hos trepigget hundestejle
The liver is dissected out and stored at – 70 °C or below.
Udtagning af vævsprøver og DNA-ekstraktion
Recommended procedures for liver excision and pre-treatment are available in OECD TG 229 (37) or Chapter C.37 of this Annex (38).
DNA kan ekstraheres ved hjælp af en række forskellige reagenser, som er tilgængelige i handlen, og både manuelle og automatiske ekstraktionssystemer.
Livers are then individually homogenised as described in OECD TG 229 or Chapter C.37 of this Annex.
Den protokol, der benyttes ved Cefas Weymouth-laboratoriet, skitseres nedenfor, og de alternative metoder er tilføjet, hvor det er relevant.
The supernatant collected is used for measuring VTG with a homologous ELISA technique (see Appendix 6 for an example of quantification in zebrafish or OECD TG 229 (37) for Japanese medaka).
Med en fin saks fjernes et lille stykke væv (10-20 mg) fra det dorsolaterale område fra hver enkelt fisk (efter at hoved og hale er fjernet til VTG-analyse).
Following this approach, it is also possible to have individual fish data on both VTG and gonad histology.
Vævet sættes i et rør og placeres enten direkte i flydende kvælstof (til opbevaring ved – 80 °C) eller fyldes med 70 % ethanol (til transport og efterfølgende opbevaring ved 4 °C).
Blood plasma method for VTG analysis
Saksen renses efter hver fisk i 70 % ethanol, herefter i destilleret vand og tørres med en papirserviet.
Blood is collected from the anaesthetised fish by cardiac puncture, caudal vein or tail cutting, and centrifuged at 4 °C for plasma collection.
Eventuelt ethanol fjernes ved bortsugning, og vævet udrådnes natten over med proteinase K i 400 μl ATL buffer (Qiagen).
The plasma is stored at – 70 °C or below until use.
En delprøve (200 μl) af spaltningsproduktet overføres til en S-blok med 96 brønde (Qiagen), og DNA'et ekstraheres i et format med 96 brønde ved hjælp af Qiagen Universal BioRobot og QIamp Investigator BioRobot kit.
The whole fish is euthanised and fixed for histology.
DNA'et elueres i 50 μl DNase- og RNasefrit vand.
Both plasma samples and fish are numbered individually to relate VTG levels to the sex of the fish.
Hvis der anvendes hårdt væv til at ekstrahere DNA (såsom en hvirvelsøjle eller en brystfinne), kan det være nødvendigt at homogenisere prøven i lysisbufferen ved hjælp af en FastPrep® tissue lyser eller tilsvarende vævsnedbrydningssystem.
How to cut a fish for measurement of VTG in head/tail homogenate and histological evaluation of the mid section
Alternativt
Genetic sex determination
Vævet udrådnes natten over med proteinase K i 400 μl G2-lysisbuffer (Qiagen), og DNA'et ekstraheres fra 200 μl af spaltningsproduktet ved hjælp af enten EZ-1 DNA easy tissue kit og EZ-1 biorobot eller DNA easy tissue mini kit.
A biological sample for the determination of the genetic sex is taken from individual fish in species possessing appropriate markers.
DNA'et elueres i en 50 μl volumen.
For Japanese medaka, the anal fin or dorsal fin is collected.
Vævene behandles med DNAzol reagens.
A detailed description is given in Appendix 9 including tissue sampling and sex determination by a PCR-method.
Vævsprøverne lyseres i 1 ml DNAzol i 10 minutter i et 1,5 ml mikrocentrifugerør og centrifugeres herefter ved 13000 rpm i 5 minutter for at fjerne eventuelle partikler.
Equally, for the three spined stickleback, a description of tissue sampling and a sex determining PCR-method is given in Appendix 10.
Den lyserede prøve overføres herefter til et nyt 1,5 ml mikrocentrifugerør indeholdende 500 μl 100 % ethanol af molekylærbiologisk kvalitet og centrifugeres ved 13000 rpm i 10 minutter for at udfælde DNA'et.
VTG measurement
Ethanolen fjernes og erstattes med 400 μl 70 % ethanol af molekylærbiologisk kvalitet, centrifugeres ved 13000 rpm i 5 minutter, og DNA-pelleten opløses i 50 μl molekylær DNase- og RNasefrit vand.
The measurement of VTG should be based upon a quantitative and analytically validated method.
Hvis der anvendes hårdt væv (brystfinne), kan det være nødvendigt at homogenisere prøven i lysisbufferen ved hjælp af en FastPrep® tissue lyser eller tilsvarende vævsnedbrydningssystem forud for ekstraheringen af DNA.
Information should be available upon the intra-assay and inter-assay variability of the method used in a given laboratory.
DNA'et opbevares ved – 20 °C, indtil det skal bruges.
The source of inter- and intra-laboratory variability is (most likely) based on the different developing stages of the fish population.
der skal bæres handsker under procedurerne.
Considering the variability of VTG measurement, NOECs based on this endpoint alone should be treated with great care.
Analyse af polymerasekædereaktion (PCR)
Different methods are available to assess VTG production in the fish species considered in this assay.
Der blev udført amplificeringer ved hjælp af 2,5 μl DNA-ekstrakt i et 50 μl reaktionsvolumen under anvendelse af Idh locus-primere (som beskrevet af Peichel et al., 2004.
A measurement technique that is both relatively sensitive and specific is the determination of protein concentrations via enzyme-linked immunosorbent assay (ELISA).
Current Biology 1:1416-1424):
Homologous antibodies (raised against VTG of the same species) and most important homologous standards should be used.
Fremadrettet primer
Sex determination
Bagudrettet primer
Dependent on the VTG sampling procedure, whole fish or the remaining mid-section of each fish is placed in a pre-labelled processing cassette and fixed in an appropriate fixative for histological determination of sex (optionally also for evaluation of gonadal staging).
Der findes mange leverandører af egnede PCR-reagenser.
Guidance on fixation and embedding is provided in Appendix 7 as well as in the OECD Guidance Document on the Diagnosis of Endocrine-Related Histopathology of Fish Gonads (22).
Fremstilling af »reaktionsblandingen« (50 μl pr. prøve):
The individuals should be placed longitudinally in the paraffin block.
En masterblanding fremstilles på følgende måde:
At least six longitudinal sections (3-5 μm in thickness) in a frontal plane including gonadal tissue from both gonads are taken from each individual.
Den kan fremstilles på forhånd og opbevares frossen ved – 20 °C, indtil den skal bruges.
The interval between these sections should be approximately 50 μm for males and 250 μm for females.
Der laves tilstrækkelig masterblanding til en negativ kontrol (kun vand af molekylærbiologisk kvalitet).
However, since each block will often contain males and females (if more than one individual are embedded in each block), the interval between sections from these blocks should be approximately 50 μm until at least six sections of the gonads from each male are obtained.
Volumen (stamkonc.)/prøve
Thereafter, the interval between sections can be increased to approximately 250 μm for the females.
Nukleotider (dATP, dCTP, dGTP, dTTP)
Sections are stained with haematoxylin and eosin and examined by light-microscopy with focus on sex (male, female, intersex or undifferentiated).
0,5 μl (25 mM hver)
Intersex is defined as presence of more than one oocyte in testis per six sections analysed or spermatogenic cells (yes/no) in ovaries.
250 μM hver
Histopathology and staging of ovaries and testis is optional but if investigated, the results should be statistically analyzed and reported.
Fremadrettet primer
It should be noted that some fish species naturally lack a fully developed pair of gonads and only one gonad may be present (e.g. Japanese medaka and occasionally zebrafish).
Bagudrettet primer
Genetic sex determination in individual Japanese medaka is based on the presence or absence of the medaka male-sex determining gene, DMY, which is located on the Y chromosome.
2,0 μM
The genotypic sex of medaka can be identified by sequencing the DMY gene from DNA extracted from for instance a piece of anal fin or dorsal fin.
Hæld 47,5 μl i et mærket 0,5 ml tyndvægget PCR-rør.
Guidance for tissue preparation and PCR method is given in Appendix 9.
Tilsæt 2,5 μl oprenset DNA til det korrekt mærkede rør.
The genetic sex determination in individual three-spined stickleback is also performed via a PCR method, described in Appendix 10.
Gentag for alle prøver og den negative kontrol.
The occurrence of intersex (for definition, see Appendix 1) should be reported.
Dryp to dråber mineralolie i overfladen.
Secondary sexual characteristics
Brug alternativt en thermocycler med et opvarmet låg.
Secondary sexual characteristics are under endocrine control in species like the Japanese medaka;
Luk lågene.
therefore observations of physical appearance of the fish should if possible be made at the end of the exposure.
Prøverne blev denatureret i en Peltier PTC-225 thermocycler ved 94 ± 2 °C i 5 minutter efterfulgt af 39 cyklusser ved 94 ± 2 °C i 1 minut, 55 ± 2 °C i 1 minut, 72 ± 2 °C i 1 minut og en endelig forlængelse på 72 ± 2 °C i 10 minutter.
In the Japanese medaka, the papillary formation on the posterior part of the anal fin in females is androgen sensitive.
Fremstilling af agarosegel (2 %):
Chapter C.37 of this Annex (38) provides relevant photographs of male secondary sex characteristics and androgenised females.
Traditionelt opløses PCR-produkter på en 20 % agarosegel indeholdende ethidiumbromid.
It is important that the strongest valid statistical test determine the endpoint.
Kapillarbaserede elektroforesesystemer kan også bruges.
The replicate is the experimental unit but intra-replicate variability should be included in the statistical testing.
Afvej 2 g agarose i 100 ml 1 × TAE-buffer.
A decision flow-chart is available in Appendix 8 to help with the most appropriate statistical test to use based on the characteristic of the data obtained from the test.
Opvarm agarosen i en mikroovn (ca. 2-3 min.), så den opløses.
Statistical significance level is 0,05 for all endpoints included.
Overfør den varme opløsning til gelstøbningsapparatet.
The proportions of sex should be analysed for significant effect (NOEC/LOEC approach) of exposure by Jonckheere-Terpstra (Trend test) if a monotone dose-response exists.
Lad gelen hærde.
If non-monotonicity is found then a pair wise test should be applied:
Gelelektroforese:
Use Dunnett's test if normality and homogenous variance can be obtained.
Overfør agarosegelen til elektroforeseapparatet og nedsænk i 1 × TAE-buffer.
Use Tamhane-Dunnett if heterogeneous variance is present.
Hæld 20 μl af hver prøve i en separat brønd og tilsæt en molekylvægtmarkør (100bp DNA ladder, Promega) til en overskydende brønd.
Otherwise use exact Mann-Whitney test with Bonferroni-Holm adjustment.
Elektroforese udføres ved 120 V i 30-45 minutter.
A flow chart describing the statistics of the proportions of sex is placed in Appendix 8.
Visualisering af amplificeringsprodukter
The proportions of sex should be presented in tables as concentration proportions ± SD of males, females, intersex and undifferentiated.
Hvis ethidiumbromidet blev inkorporeret i agarosegelen som beskrevet ovenfor, visualiseres DNA-produkterne under en UV-kilde.
Statistical significance should be highlighted.
Alternativt farves agarosegelen ved at dække gelen i en fortyndet ethidiumbromidopløsning (0,5 μg/ml i vand) i 30 minutter før visualisering.
Examples are presented in the FSDT Phase 2 validation report (42).
Tillæg 11
Genetic sex should be reported as percentage of phenotypic sex reversal of males, females, intersex and undifferentiated.
Vejledning i kunstig befrugtning for trepigget hundestejle
VTG concentrations
Formålet med dette afsnit er at beskrive, ved hvilke fremgangsmåder der kan opnås befrugtede æg fra den trepiggede hundestejle med henblik på at bruge dem i FSDT.
VTG concentrations should be analysed for significant effect (NOEC/LOEC approach) of exposure.
Fremgangsmåder
The Dunnett test is preferable to the t-test with Bonferroni correction.
Udtagning af sperm fra hannerne
Where a Bonferroni correction is used, the Bonferroni-Holm correction is preferable.
En han med en god farve fra den ønskede population aflives.
Allowance should be made for log-transformation of VTG to achieve normality and variance homogeneity.
Testiklerne fridissekeres fra hver side af fisken.
Next, if the concentration-response is consistent with monotonicity, then the JonckheereTerpstra test is preferable to any of the above.
Testiklerne er normalt stærkt pigmenterede, stavformede strukturer, som let kan ses ved kroppens laterale midterlinje.
If t-tests or Dunnett's test is used, there is no need for a ANOVA significance F-test in order to proceed.
Benyt en af følgende metoder:
For details see the flow chart in Appendix 8.
Begynd ved gattet og læg med en fin saks en 1-1,5 cm incision med et enkelt klip i en vinkel på omkring 45 grader.
Results should be reported in tables as concentration means ± SD for males, females, intersex and undifferentiated separately.
Brug en skalpel til at foretage en lille incision i fiskens side lidt bag pelvis og lige ventralt for de laterale plader.
Statistical significance for phenotypic females and phenotypic males should be highlighted.
Testiklerne fjernes ved hjælp af en fin pincet og anbringes i en petriskål.
Test chemical actual concentrations
Hver testikel dækkes med 100 μl frisklavet Hank's final solution.
The actual chamber concentrations of the test chemical should be analysed in frequencies described in paragraph 34.
Testiklerne skæres i fine terninger med et barberblad eller en skalpel.
Results should be reported in tables as mean concentration ± SD on replicate basis as well as on concentration basis with information on number of samples and with outliers from the mean treatment concentration ± 20 % highlighted.
Dette vil frigøre spermen og give Hank's solution et mælkeagtigt udseende.
Examples can be found in the FSDT Phase 2 validation report (42).
Væsken, som indeholder sperm, hældes i et rør, idet det tilstræbes ikke at medtage stykker af testikelvæv ved pipetteringen.
The test results should be interpreted with caution where measured test chemical concentrations in test solutions occur at levels near the detection limit of the analytical method.
800 μl Hank's final solution tilsættes til røret og blandes godt.
Relevant physical-chemical properties;
Om nødvendigt kan hannen præserveres ved fiksering i 100 % ethanol eller andet ønsket fikseringsmiddel.
chemical identification data including purity and analytical method for quantification of the test chemical.
Dette er særligt vigtigt, hvis undersøgelsen udpeger afkommets ophav.
Test procedure used (e.g. flow-through semi-static/renewal);
Skønt de fleste nødvendige stamopløsninger kan fremstilles i forvejen, skal stamopløsning 5 og den efterfølgende slutopløsning være frisklavet på brugsdagen.
test design including test concentrations, method of preparation of stock solutions (in an Annex), frequency of renewal (the solubilising agent and its concentration should be given, when used);
Stamopløsning 1
The nominal test concentrations, the means of the measured values and their standard deviations in the test chambers and the method by which these were attained (the analytical method used should be presented in an Annex);Evidence that the measurements refer to the concentrations of the test chemical in true solution;
Destilleret vand (DV)
Water quality within test chambers:
Stamopløsning 2
Detailed information on feeding (e.g. type of food(s), source, amount given and frequency and analyses for contaminants (e.g. PCBs, PAHs and organochlorine pesticides) if relevant.
HPO4 (vandfrit)
Evidence that controls met the validity criteria:
Stamopløsning 3
data on hatching rate should be presented in tables as percentage per replicate and per concentration.
Stamopløsning 4
Outliers from the acceptance criteria (in controls) should be highlighted.
MgSO4 · 7H2O
Survival should be presented as percentage per replicate and per concentration.
Stamopløsning 5 (frisklavet)
Outliers from the validity criteria (in controls) should be highlighted;