natgillin/translations-raw huggingface dataset:
En anden teknik til kortlægning af vorter, som er parret eller hænger sammen lodret langs mundens vandrette plan, er dobbeltmarkering af to vortevurderingspunkter i en enkelt kasse.
Therefore it is recommended that the solvent concentration, if necessary, is minimised wherever technically feasible (dependent on the physical-chemical properties of the test chemical).
Kortlægningsområder:
A flow-through test system will be used.
A — Vorter omkring øjnene.
Such a system continually dispenses and dilutes a stock solution of the test chemical (e.g. metering pump, proportional diluter, saturator system) in order to deliver a series of concentrations to the test chambers.
Kortlagt fra ryg mod bug omkring øjnenes forkant.
The flow rates of stock solutions and dilution water should be checked at intervals, preferably daily, during the test and should not vary by more than 10 % throughout the test.
Almindeligvis flere hos modne hanner i kontroller, ikke til stede hos hunner i kontroller, generelt parret (en ved hvert øje) eller enkeltvis hos hunner, der eksponeres for androgener.
Care should be taken to avoid the use of low-grade plastic tubing or other materials that may contain biologically active chemicals.
B-Vorter mellem næsebor (porer, sensorisk kanal).
When selecting the material for the flow-through system, possible adsorption of the test chemical to this material should be considered.
Normalt i par hos hanner i kontroller ved højere udviklingsniveauer (2 — forstørret eller 3 — udtalt).
Test fish should be selected from a laboratory population, preferably from a single stock, which has been acclimated for at least two weeks prior to the test under conditions of water quality and illumination similar to those used in the test.
Ikke til stede hos hunner i kontroller med en vis forekomst og udvikling hos hunner, der eksponeres for androgener.
It is important that the loading rate and stocking density (for definitions, see Appendix 1) be appropriate for the test species used (see Appendix 2).
C — Vorter umiddelbart foran næseborene, parallelt med munden.
mortalities of greater than 10 % of population in seven days: reject the entire batch;
Generelt forstørrede eller udtalte hos hanner i kontroller.
mortalities of between 5 % and 10 % of population: acclimation for seven additional days; if more than 5 % mortality during second seven days, reject the entire batch;
Til stede eller forstørrede hos mindre udviklede hanner eller androgenbehandlede hunner.
mortalities of less than 5 % of population in seven days: accept the batch.
D — Vorter parallelt langs mundlinjen.
Fish should not receive treatment for disease during the acclimation period, in the pre-exposure period, or during the exposure period.
Vurderes generelt som udviklede hos hanner i kontroller.
Pre-exposure and selection of fish
Fraværende hos hunner i kontroller, men til stede hos hunner, der eksponeres for androgener.
The one to two-week pre-exposure period is recommended with animals placed in vessels similar to the actual test.
Vortemodel:
Fish should be fed ad libitum throughout the holding period and during the exposure phase.
Numerisk score
The exposure phase is started with sexually dimorphic adult fish from a laboratory supply of reproductively mature animals (e.g. with clear secondary sexual characteristics visible as far as fathead minnow and medaka are concerned), and actively spawning.
Dato
For general guidance only (and not to be considered in isolation from observing the actual reproductive status of a given batch of fish), fathead minnows should be approximately 20 (± 2) weeks of age, assuming they have been cultured at 25 ± 2 °C throughout their lifespan.
1 – tilstedeværende
Japanese medaka should be approximately 16 (± 2) weeks of age, assuming they have been cultured at 25 ± 2 °C throughout their lifespan.
Score i alt
Zebrafish should be approximately 16 (± 2) weeks of age, assuming they have been cultured at 26 ± 2 °C throughout their lifespan.
2 – forstørret
Egg production should be assessed daily during the pre-exposure phase.
3 – udtalt
It is recommended that spawning be observed in all replicate tanks prior to inclusion in the exposure phase of the assay.
Tillæg 5B
Quantitative guidance on desirable daily egg production cannot be provided at this stage, but it is relatively common to observe average spawns of > 10 eggs/female/day for each species.
VURDERING AF SEKUNDÆRE KØNSKARAKTERISTIKA HOS JAPANSK RISFISK MED HENBLIK PÅ PÅVISNING AF VISSE HORMONFORSTYRRENDE KEMIKALIER
A randomised block design according to egg production output should be used to allocate replicates to the various experimental levels to ensure balanced distribution of replicates.
Nedenfor findes en beskrivelse af målingen af papillære strukturer [1], som er de sekundære kønskarakteristika hos japansk risfisk (Oryzias latipes).
TEST DESIGN
Efter udskæring af leveren (tillæg 6) anbringes kadaveret i et konisk rør, som indeholder ca. 10 ml 10 % neutralt bufret formalin (opad: hoved, nedad: hale).
Three concentrations of the test chemical, one control (water) and, if needed, one solvent control are used.
Hvis kønskirtlen er fikseret i andet end 10 % neutralt bufret formalin, foretages et tværsnit på tværs af kadaveret mellem den forreste del af gatfinnen og anus med en skalpel, idet det omhyggeligt undgås at skade kønsåbningen og selve kønskirtlen (figur 3).
The data may be analysed in order to determine statistically significant differences between treatment and control responses.
Anbring kraniesiden af fiskekroppen i fikseringsopløsningen for at præservere kønskirtlen og halesiden af fiskekroppen i 10 % neutralt bufret formalin som beskrevet ovenfor.
These analyses will inform whether further longer term testing for adverse effects (namely, survival, development, growth and reproduction) is required for the chemical, rather than for use in risk assessment (25).
Når fiskekroppen er anbragt i 10 % neutralt bufret formalin, gribes den forreste del af gatfinnen med en pincet og holdes bøjet i omkring 30 sekunder for at holde gatfinnen åben.
For zebrafish, on day 21 of the experiment, males and females from each treatment level (5 males and 5 females in each of the two replicates) and from the control(s) are sampled for the measurement of vitellogenin.
Når gatfinnen gribes med pincetten, tager man forsigtigt fat i nogle få finnestråler i den forreste del for ikke at ridse de papillære strukturer.
For medaka, on day 21 of the experiment, males and females from each treatment level (3 males and 3 females in each of the four replicates) and from the control(s) are sampled for the measurement of vitellogenin and secondary sex characteristics.
Når gatfinnen har været holdt åben i omkring 30 sekunder, opbevares fiskekroppen i 10 % neutralt bufret formalin ved stuetemperatur indtil måling af de papillære strukturer (måling bør foretages efter fiksering i mindst 24 timer).
For fathead minnow, on day 21 of exposure, males and females (2 males and 4 females in each of the four replicates) and from the control(s) are sampled for the measurement of vitellogenin and secondary sex characteristics.
Måling
Quantitative assessment of fecundity is required, and gonadal tissues should be fixed in whole or dissected for potential histopathological evaluation, if required.
Når fiskekroppen har været fikseret i 10 % neutralt bufret formalin i mindst 24 timer, opsamles fiskekadaveret fra det koniske rør, og formalinet tørres af med filterpapir (eller en papirserviet).
Selection of test concentrations
Anbring fisken med abdomen opad.
For the purposes of this test, the highest test concentration should be set by the maximum tolerated concentration (MTC) determined from a range finder or from other toxicity data, or 10 mg/l, or the maximum solubility in water, whichever is lowest.
Afskær derefter forsigtigt gatfinnen med en lille dissektionssaks (det foretrækkes, at en smule pterygiophor afskæres sammen med gatfinnen).
The MTC is defined as the highest test concentration of the chemical which results in less than 10 % mortality.
Grib den forreste del af den afskårne gatfinne med en pincet, og anbring den på en glasplade med flere dråber vand.
Using this approach assumes that there are existing empirical acute toxicity data or other toxicity data from which the MTC can be estimated.
Dæk derefter gatfinnen med et dækglas.
Estimating the MTC can be inexact and typically requires some professional judgment.
Det er vigtigt ikke at ridse de papillære strukturer, når man griber gatfinnen med pincetten.
Three test concentrations, spaced by a constant factor not exceeding 10, and a dilution-water control (and solvent control if necessary) are required.
Tæl antallet af ledskiver med papillære strukturer ved hjælp af tælleren i et biologisk mikroskop (opretstående mikroskop eller omvendt mikroskop).
A range of spacing factors between 3,2 and 10 is recommended.
De papillære strukturer genkendes, når en lille formation af strukturer er synlig på bagkanten af ledskiven.
Selection and weighing of test fish
Registrer antallet af ledskiver med papillære strukturer i hver finnestråle på arbejdsarket (f.eks. første finnestråle: 0, anden finnestråle: 10, tredje finnestråle: 12 osv.), og indtast summen i Excel-arket for hver fisk.
It is important to minimise variation in weight of the fish at the beginning of the assay.
Tag om nødvendigt et billede af gatfinnen, og tæl antallet af ledskiver med papillære strukturer på billedet.
Suitable size ranges for the different species recommended for use in this test are given in Appendix 2.
Efter målingen anbringes gatfinnen i det koniske rør, der er beskrevet i (1), og opbevares.
For the whole batch of fish used in the test, the range in individual weights for male and female fish at the start of the test should be kept, if possible, within ± 20 % of the arithmetic mean weight of the same sex.
Figur 1.
It is recommended to weigh a subsample of the fish stock before the test in order to estimate the mean weight.
Diagram, der viser kønsforskelle i gatfinnens form og størrelse.
The test duration is 21 days, following a pre-exposure period.
A, han; B, hun.
The recommended pre-exposure period is one to two weeks.
Figur 2.
Fish should be fed ad libitum with an appropriate food (Appendix 2) at a sufficient rate to maintain body condition.
A — Strukturer på gatfinnes ledskiver.
Care should be taken to avoid microbial growth and water turbidity.
J.P.
As a general guidance, the daily ration may be divided into two or three equal portions for multiple feeds per day, separated by at least three hours between each feed.
(joint plate): ledskive; A.S. (axial space): aksial afstand; P. (process): struktur.
A single larger ration is acceptable particularly for weekends.
B — Finnestråles distale ekstremitet.
Food should be withheld from the fish for 12 hours prior to sampling/necropsy.
Actinotrichia (Act.) er på spidsen.
Fish food should be evaluated for the presence of contaminants such as organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs).
Figur 3.
Food with an elevated level of phytoestrogens that would compromise the response of the assay to known oestrogen agonist (e.g. 17β- estradiol) should be avoided.
Billede af fiskekrop, der viser det sted, hvor der skal skæres, når kønskirtlen er fikseret i en anden fikseringsopløsning end 10 % neutralt bufret formalin.
Uneaten food and faecal material should be removed from the test vessels at least twice weekly, e.g. by carefully cleaning the bottom of each tank using a siphon.
I givet fald afskæres den resterende del af kroppen mellem den forreste del af gatfinnen og anus med en skalpel (rød streg).
Prior to initiation of the exposure period, proper function of the chemical delivery system should be ensured.
Hovedsiden af fiskens krop anbringes i fikseringsopløsningen til kønskirtlen, og halesiden af fiskekroppen anbringes i 10 % neutralt bufret formalin.
All analytical methods needed should be established, including sufficient knowledge on the chemical stability in the test system.
Papillære strukturer ses normalt kun hos voksne hanner og observeres på finnestråler fra den anden til den syvende eller ottende talt fra den bageste ende af gatfinnen (figur 1 og 2).
During the test, the concentrations of the test chemical are determined at regular intervals, as follows: the flow rates of diluent and toxicant stock solution should be checked preferably daily but as a minimum twice per week, and should not vary by more than 10 % throughout the test.
Strukturerne forekommer dog sjældent på den første finnestråle fra den bageste ende af gatfinnen.
It is recommended that the actual test chemical concentrations be measured in all vessels at the start of the test and at weekly intervals thereafter.
Denne standardprocedure omfatter måling af strukturer på den første finnestråle (i denne standardprocedure henviser finnestrålenummeret til rækkefølgen fra den bageste ende af gatfinnen).
It is recommended that results be based on measured concentrations.
ANBEFALEDE PROCEDURER FOR PRØVEUDTAGNING TIL VITELLOGENINANALYSE
However, if 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.
Det sikres, at der ikke forekommer krydskontamination mellem VTG-prøver fra hanner og hunner.
Samples may need to be filtered (e.g. using a 0,45 μm pore size) or centrifuged.
Fremgangsmåde 1A:
If needed, then centrifugation is the recommended procedure.
Tykhovedet elritse, indsamling af blod fra halevenen/-arterien
However, if the test material does not adsorb to filters, filtration may also be acceptable.
Efter bedøvelse afskæres haleroden delvist med en skalpel, og blod indsamles fra halevenen/-arterien med et hepariniseret mikrohæmatokrit-kapillærrør.
During the test, dissolved oxygen, temperature, and pH should be measured in all test vessels at least once per week.
Når blodet er indsamlet, isoleres plasma hurtigt ved centrifugering i 3 minutter ved 15000 g (eller alternativt i 10 minutter ved 15000 g ved 4 °C).
Total hardness and alkalinity should be measured in the controls and one vessel at the highest concentration at least once per week.
Plasmadelen fjernes derefter fra mikrohæmatokritrøret og opbevares i et centrifugeglas med 0,13 enheder aprotinin (en proteaseinhibitor) ved – 80 °C, indtil VTG kan bestemmes.
The daily quantitative monitoring of fecundity is required.
Afhængigt af størrelsen af den tykhovedede elritse (som er kønsbestemt) kan der generelt indsamles et plasmavolumen på 5-60 mikroliter pr. fisk (Jensen et al., 2001).
Measurement and evaluation of these endpoints and their utility are described below.
Fremgangsmåde 1B:
Survival
Tykhovedet elritse, indsamling af blod fra hjertet
Fish should be examined daily during the test period and any mortality should be recorded and the dead fish removed as soon as possible.
Alternativt kan blod indsamles ved hjertepunktur med en hepariniseret kanyle (1000 enheder heparin pr. ml).
Dead fish should not be replaced in either the control or treatment vessels.
Blodet overføres til Eppendorf-rør (holdt på is) og centrifugeres (5 min., 7000 g, stuetemperatur).
Sex of fish that die during the test should be determined by macroscopic evaluation of the gonads.
Plasmaet overføres til rene Eppendorf-rør (i alikvoter, hvis plasmavolumenet tillader det) og nedfryses omgående ved – 80 °C, indtil det skal analyseres (Panter et al., 1998).
Behaviour and appearance
Fremgangsmåde 2A:
Any abnormal behaviour (relative to controls) should be noted; this might include signs of general toxicity including hyperventilation, uncoordinated swimming, loss of equilibrium, and atypical quiescence or feeding.
Testfisk fjernes fra testkammeret med et lille fiskenet.
Such behavioural observations may also provide useful qualitative information to inform potential future fish testing requirements.
Derudover bør alle hanner fjernes fra et testkammer, før de resterende hunner fjernes.
Experience to date with fathead minnows suggests that some endocrine active chemicals may initially induce changes in the following external characteristics: body colour (light or dark), coloration patterns (presence of vertical bands), and body shape (head and pectoral region).
Kontroller, at etiketterne på testkammeret og transportbeholderen er korrekte, og at antallet af fisk, der er blevet fjernet fra testkammeret, og antallet af fisk, som er tilbage i testkammeret, er som forventet.
Daily quantitative observations of spawning should be recorded on a replicate basis.
I givet fald identificeres kønnet ved observation af kønskirtlen eller sekundære kønskarakteristika under stereoskopisk mikroskop.
Eggs will be removed daily from the test chambers.
Når testfisken er bedøvet, overføres den til filterpapir (eller en papirserviet) med en almindelig pincet.
It is not considered necessary to provide spawning substrate for medaka.
Anbring testfisken med abdomen opad på papirservietten (petriskål af glas eller objektglas er også en mulighed).
Such gross morphological observations may provide useful qualitative and quantitative information to inform potential future fish testing requirements.
Blotlæg den forbundne del af leveren og galdeblæren med en anden præcisionspincet.
Vitellogenin (VTG)
Sørg for ikke at ødelægge galdeblæren.
Depending upon the size of the fish, collectable blood volumes generally range from 5 to 60 μl per individual for fathead minnows and 5-15 μl per individual for zebrafish.
Fjern fedtmassen og andet væv fra leverens periferi.
It is recommended to use a method capable to detect VTG levels as low as few ng/ml plasma (or ng/mg tissue), which is the background level in unexposed male fish.
Anbring leveren på objektglasset.
Each ELISA plate used for VTG assays should include the following quality control samples: at least 6 calibration standards covering the range of expected VTG concentrations, and at least one non-specific binding assay blank (analysed in duplicate).
Bekræft identifikationsinformationen på mikrorørets etiket.
The correlation coefficient (R2) for calibration curves should be greater than 0,99.
Luk låget på det mikrorør, der indeholder leveren.
However, a high correlation is not sufficient to guarantee adequate prediction of concentration in all ranges.
Opbevar det på et kølestativ (eller isstativ).
In addition to having a sufficiently high correlation for the calibration curve, the concentration of each standard, as calculated from the calibration curve, should all fall between 70 and 120 % of its nominal concentration.
Efter udskæringen af en lever rengøres dissektionsinstrumenterne eller erstattes med rene instrumenter.
If the nominal concentrations trend away from the calibration regression line (e.g. at lower concentrations), it may be necessary to split the calibration curve into low and high ranges or to use a nonlinear model to adequately fit the absorbance data.
Fjern leverne fra alle fiskene i transportbeholderen som beskrevet ovenfor.
If the curve is split, both line segments should have R2 > 0,99.
Når leverne er skåret ud af alle fisk i transportbeholderen (dvs. alle hanner eller hunner i et testkammer), anbringes alle leverprøver i et reagensglasstativ med en identifikationsetiket og opbevares i en fryser.
The limit of detection (LOD) is defined as the concentration of the lowest analytical standard, and limit of quantitation (LOQ) is defined as the concentration of the lowest analytical standard multiplied by the lowest dilution factor.
Når leverne gives til forbehandling kort efter udskæringen, bæres prøverne til den næste arbejdsstation på et kølestativ (eller isstativ).
On each day that VTG assays are performed, a fortification sample made using an inter-assay reference standard will be analysed (Appendix 7).
Efter udskæring af leveren kan fiskekadaveret anvendes til histologisk undersøgelse af kønskirtlerne og måling af sekundære kønskarakteristika.
The ratio of the expected concentration to the measured concentration will be reported along with the results from each set of assays performed on that day.
Prøveemne
Evaluation of gonadal histopathology
Der foretages et snit lige foran brystfinnerne med en saks.
In this respect, gonads are fixed either whole body or dissected.
Der foretages et snit langs midterlinjen af abdomen til et punkt ca. 2 mm foran anus.
When histopathology is required, specific endocrine-related responses on the gonads will be looked for in the assessment of the endocrine activity of the test chemical.
Bugvæggene spredes med pincet for at blotlægge leveren og andre indre organer.
These diagnostic responses essentially include the presence of testicular oocytes, Leydig cell hyperplasia, decreased yolk formation, increased spermatogonia and perifollicular hyperplasia.
(Alternativt hæftes bugvæggene op lateralt).
Other gonadal lesions like oocyte atresia, testicular degeneration, and stage changes, may have various causes.
Pilen angiver leveren.
The Guidance document on fish gonadal histopathology specifies procedures that will be used in the dissection, fixation, sectioning and histopathological evaluation of the gonads (22).
Indvoldene tages forsigtigt ud med pincet.
Where a solvent control is used, an appropriate statistical test should be performed between the dilution water and solvent controls for each endpoint.
Figur 6
Guidance on how to handle dilution water and solvent control data in the subsequent statistical analysis can be found in OECD, 2006c (28).
Begge ender af indvoldene og eventuelle tilknyttede mesenteriske strukturer afskæres med saks.
All biological response data should be analysed and reported separately by sex.
Figur 7 (hun)
If the required assumptions for parametric methods are not met — non-normal distribution (e.g. Shapiro-Wilk's test) or heterogeneous variance (Bartlett's test or Levene's test), consideration should be given to transforming the data to homogenise variances prior to performing the ANOVA, or to carrying out a weighted ANOVA.
Proceduren er identisk for hundyr.
Dunnett's test (parametric) on multiple pair-wise comparisons or a Mann-Whitney with Bonferroni adjustment (non-parametric) may be used for non-monotonous dose-response.
Figur 8
Other statistical tests may be used (e.g. Jonckheere-Terpstra test or Williams test) if the dose-response is approximately monotone.
Den afsluttede procedure.
A statistical flowchart is provided in Appendix 8 to help in the decision on the most appropriate statistical test to be used.
Japansk risfisk (Oryzias latipes), forbehandling af lever med henblik på vitellogeninanalyse
Reporting of test results
Tag kolben med homogenatbuffer fra ELISA-kittet, og afkøl den med knust is (opløsningens temperatur: ≤ 4 °C).
Study data should include:
Hvis der bruges homogenatbuffer fra EnBio ELISA-systemet, optøs opløsningen ved stuetemperatur, og kolben afkøles med knust is.
Testing facility:
Klargøring af leveren til forbehandling
Physical nature and relevant physicochemical properties
Antallet af 1,5 ml-mikrorør, som indeholder leverprøver taget fra fryseren, må på et givet tidspunkt ikke overstige det antal, der kan centrifugeres på det pågældende tidspunkt.
Justification of choice of solvent (if other than water)
Arranger 1,5 ml-mikrorørene med leverprøverne i samme rækkefølge som prøvenummeret på isstativet (det er ikke nødvendigt at optø leveren).
Species and strain
Udførelse af forbehandlingen
Supplier and specific supplier facility
Tilsætning af homogenatbufferen
Age of the fish at the start of the test and reproductive/spawning status
Se på listen, hvilket homogenatbuffervolumen der skal bruges til en specifik leverprøve, og juster mikropipetten (volumeninterval: 100-1000 μl) til det rette volumen.
Details of animal acclimation procedure
Sæt en ren spids på mikropipetten.
Body weight of the fish at the start of the exposure (from a sub-sample of the fish stock)
Tag homogenatbufferen fra reagensglasset, og tilsæt bufferen til 1,5 ml-mikrorøret med leveren.
Test procedure used (test-type, loading rate, stocking density, etc.);
Tilsæt homogenatbuffer til alle 1,5 ml-mikrorør med lever i henhold til ovennævnte procedure.
Method of preparation of stock solutions and flow-rate;