natgillin/translations-raw huggingface dataset:
Tillæg 5A
Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, and Ni), major anions and cations (e.g. Ca2+, Mg2+, Na+, K+, Cl-, and SO42-), pesticides (e.g. total organophosphorus and total organochlorine pesticides), total organic carbon and suspended solids should be made, for example, every three months where dilution water is known to be relatively constant in quality.
Vurdering af sekundære kønskarakteristika hos tykhovedet elritse med henblik på påvisning af visse hormonforstyrrende kemikalier
If water quality has been demonstrated to be constant over at least one year, determinations can be less frequent and intervals extended (e.g. every six months).
Oversigt
Some chemical characteristics of acceptable dilution water are listed in Appendix 3.
Potentielt vigtige udseenderelaterede karakteristika hos voksne fisk af arten tykhovedet elritse i test til påvisning af hormonforstyrrende stoffer omfatter kropsfarve (lys/mørk), farvemønstre (tilstedeværelse eller fravær af lodrette bånd), kropsform (hoved- og brystområde, udvidet abdomen) og specifikke sekundære kønskarakteristika (antal og størrelse af legevorter, størrelse af nakkefremspring og æglægningsorgan).
Test solutions of the chosen concentrations are prepared by dilution of a stock solution.
Legevorter befinder sig på hovedet (nakkefremspringet) hos formeringsaktive tykhovedet elritse-hanner og er sædvanligvis arrangeret i et bilateralt symmetrisk mønster (Jensen et al., 2001).
The stock solution should preferably be prepared by simply mixing or agitating the test chemical in dilution water by using mechanical means (e.g. stirring or ultrasonication).
Hunner og afkom af hankøn og hunkøn i kontrollerne udvikler ikke vorter (Jensen et al., 2001).
Saturation columns (solubility columns) can be used for achieving a suitable concentrated stock solution.
Der kan være op til otte individuelle vorter omkring hannernes øjne og mellem næseborene.
The use of a solvent carrier is not recommended.
Det største antal og de største vorter befinder sig i to parallelle linjer umiddelbart under næseborene og omkring munden.
However, in case a solvent is necessary, a solvent control should be run in parallel, at the same solvent concentration as the chemical treatments.
Hos mange fisk er der grupper af vorter under underkæben;
For difficult test chemicals, a solvent may be technically the best solution;
dem, der er tættest på munden, forekommer som regel som et enkelt par, mens sættet tættere på bugen kan bestå af op til fire vorter.
the OECD Guidance Document on aquatic toxicity testing of difficult substances and mixtures should be consulted (22).
Det faktiske antal vorter er sjældent højere end 30 (interval: 18-28; Jensen et al., 2001).
The choice of solvent will be determined by the chemical properties of the chemical.
De fremherskende vorter (for så vidt angår antal) er til stede som en enkelt, relativt rund struktur med en højde omtrent svarende til radius.
The OECD Guidance Document recommends a maximum of 100 μl/l, which should be observed.
De fleste formeringsaktive hanner har også, i det mindste nogle, vorter, som er så forstørrede og fremtrædende, at de ikke kan skelnes som individuelle strukturer.
However a recent review (23) highlighted additional concerns when using solvents for endocrine activity testing.
Visse typer hormonforstyrrende kemikalier kan forårsage abnormal forekomst af visse sekundære kønskarakteristika hos det modsatte køn;
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).
androgenreceptoragonister, såsom methyltestosteron-17β eller trenbolon-17β, kan f.eks. få tykhovedet elritse-hunner til at udvikle legevorter (Smith, 1974; Ankley et al., 2001; 2003), mens østrogenreceptoragonister kan mindske antallet eller størrelsen af legevorter hos hanner (Miles-Richardson et al., 1999; Harries et al., 2000).
A flow-through test system will be used.
Specifikke produkter og/eller specifikt udstyr kan erstattes med tilgængelige sammenlignelige alternativer.
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.
Observation opnås bedst med et forstørrelsesglas med lys eller et 3X-dissektionsmikroskop med lys.
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.
Observer fisken fra ryggen og med forenden fremad (hoved mod objektiv).
Care should be taken to avoid the use of low-grade plastic tubing or other materials that may contain biologically active chemicals.
Anbring fisken i en lille petriskål (f.eks. 100 mm i diameter) med forenden fremad og bugen nedad.
When selecting the material for the flow-through system, possible adsorption of the test chemical to this material should be considered.
Fokuser med søgeren for at identificere vorterne.
Holding of fish
Rul forsigtigt og langsomt fisken fra side til side for at identificere områder med vorter.
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.
Tæl og vurder vorterne.
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).
Gentag observationen for den ventrale overflade af hovedet ved at anbringe fisken på ryggen med forenden fremad i petriskålen.
Following a 48-hour settling-in period, mortalities are recorded and the following criteria applied:
Observationer skal være afsluttet inden for to minutter pr. fisk.
mortalities of greater than 10 % of population in seven days:
Tælling og vurdering af vorter
reject the entire batch;
Der er identificeret seks områder for vurdering af tilstedeværelsen og udviklingen af vorter hos voksen tykhovedet elritse.
mortalities of between 5 % and 10 % of population:
Der er udviklet en model for kortlægning af placering af og antal vorter (se sidst i dette tillæg).
acclimation for seven additional days;
Antallet af vorter registreres, og deres størrelse kan kvantitativt vurderes som følger:
if more than 5 % mortality during second seven days, reject the entire batch;
0 — fraværende, 1 — tilstedeværende, 2 — forstørret og 3 — udtalt for hver organisme (figur 1).
mortalities of less than 5 % of population in seven days:
Score 0 — fravær af vorter.
accept the batch
Score 1 — tilstedeværelse af vorter; identificeres som vorter med et enkelt punkt, hvis højde stort set svarer til dets radius (diameter).
Fish should not receive treatment for disease during the acclimation period, in the pre-exposure period, or during the exposure period.
Score 2 — forstørret vorte; identificeres som væv, der ligner en asterisk, normalt med en stor radial base med fordybninger ud fra midten.
Pre-exposure and selection of fish
Vorten er ofte mere forrevet, men kan undertiden være en smule afrundet.
A one-week pre-exposure period is recommended, with animals placed in vessels similar to the actual test.
Score 3 — udtalt vorte; er normalt ret stor og afrundet med mindre defineret struktur.
Fish should be fed ad libitum throughout the holding period and during the exposure phase.
Vorterne løber nogle gange sammen og danner en enkelt masse i et område eller en kombination af områder (B, C og D, beskrevet nedenfor).
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.
Farve og mønster ligner score 2, men kan være forholdsvis vilkårligt.
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.
Med dette vurderingssystem opnås normalt en samlet score for vorter på < 50 hos en normal han i kontroller med 18-20 vorter (Jensen et al., 2001).
Japanese medaka should be approximately 16 (± 2) weeks of age, assuming they have been cultured at 25 ± 2 °C throughout their lifespan.
Yderligere vurderingstal kan i givet fald markeres i, til højre for eller til venstre for kassen.
TEST DESIGN
Modellen skal således ikke nødvendigvis være symmetrisk.
Three concentrations of the test chemical, one control (water) and, if needed, one solvent control are used.
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.
The data may be analysed in order to determine statistically significant differences between treatment and control responses.
Kortlægningsområder:
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 (24).
A Vorter omkring øjnene.
For zebrafish and medaka, 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 and secondary sex characteristics, where applicable.
Kortlagt fra ryg mod bug omkring øjnenes forkant.
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.
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.
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.
B Vorter mellem næsebor (porer, sensorisk kanal).
The MTC is defined as the highest test concentration of the chemical which results in less than 10 % mortality.
Normalt i par hos hanner i kontroller ved højere udviklingsniveauer (2 — forstørret eller 3 — udtalt).
Using this approach assumes that there are existing empirical acute toxicity data or other toxicity data from which the MTC can be estimated.
Ikke til stede hos hunner i kontroller med en vis forekomst og udvikling hos hunner, der eksponeres for androgener.
Estimating the MTC can be inexact and typically requires some professional judgment.
C Vorter umiddelbart foran næseborene, parallelt med munden.
Three test concentrations, spaced by a constant factor not exceeding 10, and a dilution-water control (and solvent control if necessary) are required.
Generelt forstørrede eller udtalte hos hanner i kontroller.
A range of spacing factors between 3,2 and 10 is recommended.
Til stede eller forstørrede hos mindre udviklede hanner eller androgenbehandlede hunner.
Selection and weighing of test fish
D Vorter parallelt langs mundlinjen.
It is important to minimise variation in weight of the fish at the beginning of the assay.
Vurderes generelt som udviklede hos hanner i kontroller.
Suitable size ranges for the different species recommended for use in this test are given in Appendix 2.
Fraværende hos hunner i kontroller, men til stede hos hunner, der eksponeres for androgener.
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.
Numerisk score
It is recommended to weigh a subsample of the fish stock before the test in order to estimate the mean weight.
Dato
Conditions of exposure
Dato:
The test duration is 21 days, following a pre-exposure period.
Score i alt
The recommended pre-exposure period is one week.
3 — udtalt
Fish should be fed ad libitum with an appropriate food (Appendix 2) at a sufficient rate to maintain body condition.
Tillæg 5b
Care should be taken to avoid microbial growth and water turbidity.
Vurdering af sekundære kønskarakteristika hos japansk risfisk med henblik på påvisning af visse hormonforstyrrende kemikalier
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.
Nedenfor findes en beskrivelse af målingen af papillære strukturer, som er de sekundære kønskarakteristika hos japansk risfisk (Oryzias latipes).
A single larger ration is acceptable particularly for weekends.
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).
Food should be withheld from the fish for 12 hours prior to sampling/necropsy.
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).
Fish food should be evaluated for the presence of contaminants such as organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs).
Anbring kraniesiden af fiskekroppen i fikseringsopløsningen for at præservere kønskirtlen og halesiden af fiskekroppen i 10 % neutralt bufret formalin som beskrevet ovenfor.
Food with an elevated level of phytoestrogens that would compromise the response of the assay to known oestrogen agonist (e.g. 17-beta estradiol) should be avoided.
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.
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.
Når gatfinnen gribes med pincetten, tager man forsigtigt fat i nogle få finnestråler i den forreste del for ikke at ridse de papillære strukturer.
The photoperiod and water temperature should be appropriate for the test species (see Appendix 2).
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).
Frequency of analytical determinations and measurements
Måling
Prior to initiation of the exposure period, proper function of the chemical delivery system should be ensured.
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).
All analytical methods needed should be established, including sufficient knowledge on the chemical stability in the test system.
Anbring fisken med abdomen opad.
During the test, the concentrations of the test chemical are determined at regular intervals, as follows:
Afskær derefter forsigtigt gatfinnen med en lille dissektionssaks (det foretrækkes, at en smule pterygiophor afskæres sammen med gatfinnen).
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.
Grib den forreste del af den afskårne gatfinne med en pincet, og anbring den på en glasplade med flere dråber vand.
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.
Dæk derefter gatfinnen med et dækglas.
It is recommended that results be based on measured concentrations.
Det er vigtigt ikke at ridse de papillære strukturer, når man griber gatfinnen med pincetten.
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.
Tæl antallet af ledskiver med papillære strukturer ved hjælp af tælleren i et biologisk mikroskop (opretstående mikroskop eller omvendt mikroskop).
Samples may need to be filtered (e.g., using a 0,45 μm pore size) or centrifuged.
De papillære strukturer genkendes, når en lille formation af strukturer er synlig på bagkanten af ledskiven.
If needed, then centrifugation is the recommended procedure.
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.
However, if the test material does not adsorb to filters, filtration may also be acceptable.
Tag om nødvendigt et billede af gatfinnen, og tæl antallet af ledskiver med papillære strukturer på billedet.
During the test, dissolved oxygen, temperature, and pH should be measured in all test vessels at least once per week.
Efter målingen anbringes gatfinnen i det koniske rør, der er beskrevet i (1), og opbevares.
Total hardness and alkalinity should be measured in the controls and one vessel at the highest concentration at least once per week.
Figur 1.
Temperature should preferably be monitored continuously in at least one test vessel.
Diagram, der viser kønsforskelle i gatfinnens form og størrelse.
A number of general (e.g. survival) and core biological responses (e.g. vitellogenin levels) are assessed over the course of the assay or at termination of the assay.
A, han;
Measurement and evaluation of these endpoints and their utility are described below.
B, hun.
Survival
Figur 2.
Fish should be examined daily during the test period and any mortality should be recorded and the dead fish removed as soon as possible.
A — Strukturer på gatfinnes ledskiver.
Dead fish should not be replaced in either the control or treatment vessels.
J.P.
Sex of fish that die during the test should be determined by macroscopic evaluation of the gonads.
(joint plate): ledskive; A.S.
Behaviour and appearance
(axial space): aksial afstand;
Any abnormal behaviour (relative to controls) should be noted;
P. (process):
this might include signs of general toxicity including hyperventilation, uncoordinated swimming, loss of equilibrium, and atypical quiescence or feeding.
struktur.
Additionally external abnormalities (such as haemorrhage, discoloration) should be noted.
B — Finnestråles distale ekstremitet.
Such signs of toxicity should be considered carefully during data interpretation since they may indicate concentrations at which biomarkers of endocrine activity are not reliable.
Actinotrichia (Act.) er på spidsen.
Such behavioural observations may also provide useful qualitative information to inform potential future fish testing requirements.
Figur 3.
For example, territorial aggressiveness in normal males or masculinised females has been observed in fathead minnows under androgenic exposure;
Billede af fiskekrop, der viser det sted, hvor der skal skæres, når kønskirtlen er fikseret i en anden fikseringsopløsning end 10 % neutralt bufret formalin.
in zebrafish, the characteristic mating and spawning behaviour after the dawn onset of light is reduced or hindered by oestrogenic or anti-androgenic exposure.
I givet fald afskæres den resterende del af kroppen mellem den forreste del af gatfinnen og anus med en skalpel (rød streg).
Because some aspects of appearance (primarily colour) can change quickly with handling, it is important that qualitative observations be made prior to removal of animals from the test system.
Hovedsiden af fiskens krop anbringes i fikseringsopløsningen til kønskirtlen, og halesiden af fiskekroppen anbringes i 10 % neutralt bufret formalin.
Experience to date with fathead minnows suggests that some endocrine active chemicals may initially induce changes in the following external characteristics:
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).
body colour (light or dark), coloration patterns (presence of vertical bands), and body shape (head and pectoral region).
Strukturerne forekommer dog sjældent på den første finnestråle fra den bageste ende af gatfinnen.
Therefore observations of physical appearance of the fish should be made over the course of the test, and at conclusion of the study
Denne standardprocedure omfatter måling af strukturer på den første finnestråle (i denne standardprocedure henviser finnestrålenummeret til rækkefølgen fra den bageste ende af gatfinnen).
Humane killing of fish
Anbefalede procedurer for prøveudtagning til vitellogeninanalyse
At day 21, i.e. at termination of the exposure, the fish should be euthanized with appropriate amounts of Tricaine (Tricaine methane sulfonate, Metacain, MS-222 (CAS 886-86-2), 100-500 mg/l buffered with 300 mg/l NaHCO3 (sodium bicarbonate, CAS 144-55-8) to reduce mucous membrane irritation; blood or tissue is then sampled for vitellogenin determination, as explained in the Vitellogenin section.
Det sikres, at der ikke forekommer krydskontamination mellem VTG-prøver fra hanner og hunner.
Observation of secondary sex characteristics
Fremgangsmåde 1A:
Some endocrine active chemicals may induce changes in specialised secondary sex characteristics (number of nuptial tubercles in male fathead minnow, papillary processes in male medaka).
Tykhovedet elritse, indsamling af blod fra halevenen/-arterien
Notably, chemicals with certain modes of action may cause abnormal occurrence of secondary sex characteristic in animals of the opposite sex;
Efter bedøvelse afskæres haleroden delvist med en skalpel, og blod indsamles fra halevenen/-arterien med et hepariniseret mikrohæmatokrit-kapillærrør.
for example, androgen receptor agonists, such as trenbolone, methyltestosterone and dihydrotestosterone, can cause female fathead minnows to develop pronounced nuptial tubercles or female medaka to develop papillary processes (11, 20, 21).
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).
It also has been reported that oestrogen receptor agonists can decrease nuptial tubercle numbers and size of the dorsal nape pad in adult males (25, 26).
Om ønsket kan hæmatokritprocenten bestemmes efter centrifugering.
Such gross morphological observations may provide useful qualitative and quantitative information to inform potential future fish testing requirements.
Plasmadelen fjernes derefter fra mikrohæmatokritrøret og opbevares i et centrifugeglas med 0,13 enheder aprotinin (en proteaseinhibitor) ved – 80 °C, indtil vitellogenin kan bestemmes.
The number and size of nuptial tubercles in fathead minnow and papillary processes in medaka can be quantified directly or more practically in preserved specimens.
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).
Recommended procedures for the evaluation of secondary sex characteristics in fathead minnow and medaka are available from Appendix 5A and Appendix 5B, respectively.
Fremgangsmåde 1B:
Vitellogenin (VTG)
Tykhovedet elritse, indsamling af blod fra hjertet
Blood is collected from the caudal artery/vein with a heparinised microhematocrit capillary tubule, or alternatively by cardiac puncture with a syringe.
Alternativt kan blod indsamles ved hjertepunktur med en hepariniseret kanyle (1000 enheder heparin pr. ml).
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.