natgillin/translations-raw huggingface dataset:
At test termination, all fish are euthanised humanely.
Det är valfritt att utföra histopatologisk analys och stadiebestämning av ovarier och testiklar, men om dessa undersökningar utförs bör resultaten analyseras och redovisas statistiskt.
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;
Det bör noteras att vissa fiskarter av naturen saknar ett fullt utvecklat par könskörtlar och att endast en könskörtel kan förekomma (t.ex. japansk risfisk och ibland sebrafisk).
optionally, histopathology (e.g. staging of gonads, severity of intersex) can be performed.
Alla sådana observationer ska registreras.
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).
Bestämning av genetiskt kön för enskilda individer av japansk risfisk bygger på förekomsten eller avsaknaden av risfiskens hanliga könsbestämmande gen, DMY, som finns på Y-kromosomen.
An overview of relevant test conditions specific for validated species:
Risfiskarnas genetiska kön kan fastställas genom att man sekvenserar DMY-genen från DNA som t.ex. extraherats från en bit av analfenan eller ryggfenan.
Japanese medaka, the three-spined stickleback and zebrafish is provided in Appendix 2.
Förekomsten av DMY visar på en XY-individ (hane) oavsett fenotyp, och avsaknaden av DMY visar på en XX-individ (hona) oavsett fenotyp (23).
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.
Vägledning för vävnadsberedning och PCR-metoden finns i tillägg 9.
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.
Bestämningen av det genetiska könet i enskilda storspigg utförs också med en PCR-metod som beskrivs i tillägg 10.
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).
Förekomsten av intersexualitet (för definition, se tillägg 1) bör rapporteras.
Test acceptance criteria
Sekundära könskaraktärer
The dissolved oxygen concentration should be at least 60 per cent of the air saturation value (ASV) throughout the test;
Hos japansk risfisk är den vårtliknande bildningen på bakre delen av honans analfena androgenkänslig.
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);
I kapitel C.37 i denna bilaga (38) finns relevanta fotografier av hanens sekundära könskaraktärer och av androgeniserade honor.
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;
Det är viktigt att man använder de mest kraftfulla giltiga statistiska testerna för att fastställa endpoints.
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;
Den experimentella enheten är replikatet, men variationer mellan replikaten bör ingå i den statistiska testningen.
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):
Ett flödesschema över beslutsgången finns i tillägg 8 för att hjälpa till med att finna det mest ändamålsenliga statistiska testet på grundval av egenskaperna hos de data som erhållits från testet.
Japanese medaka
Den statistiska signifikansnivån är 0,05 för alla endpoints som ingår.
Three-spined stickleback
Könsförhållanden och genetiskt kön
Growth
Förhållandet mellan könen bör analyseras med avseende på signifikanta effekter (NOEC/LOEC-metoden) av exponeringen med Jonckheere-Terpstras (trendtest) om det föreligger en monoton dos-respons.
Fish wet weight, blotted dry
Om icke-monotonicitet föreligger ska ett parvist test användas:
> 75 mg
Använd i annat fall Mann-Whitneys exakta test med Bonferroni-Holm-justering.
Length (standard length)
Ett flödesschema över statistiken rörande förhållandet mellan könen finns i tillägg 8.
> 20 mm
Förhållandet mellan könen bör presenteras i tabeller som koncentrationsandelar ± standardavvikelser för hanar, honor, intersexuella och odifferentierade.
> 14 mm
Den statistiska signifikansen bör lyftas fram.
Sex ratio (% males or females)
Exempel ges i valideringsrapporten för testet på fiskars könsutveckling fas 2 (42).
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.
Genetiskt kön bör rapporteras som en procentandel av de fenotypiska könsbytena hos hanar, honor, intersexuella och odifferentierade.
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.
Koncentrationer av VTG
Test chambers
Koncentrationerna av VTG bör analyseras med avseende på signifikant effekt (NOEC/LOEC-metoden) av exponeringen.
Any glass, stainless steel or other chemically inert chambers can be used.
Det är bättre att använda Dunnetts test än t-testet med Bonferronikorrigering.
The dimensions of the chambers should be large enough to allow compliance with loading rate criteria given below.
Om en Bonferronikorrigering används är Bonferroni-Holm-korrigeringen att föredra.
It is desirable that test chambers be randomly positioned in the test area.
Man bör räkna med att en logaritmisk transformation av VTG måste göras för att uppnå normalitet och varianshomogenitet.
A randomised block design with each concentration being present in each block is preferable to a completely randomised design.
Om koncentration-responsen är förenlig med monotoniciteten är Jonckheere-Terpstras test att föredra framför något av de ovanstående.
The test chambers should be shielded from unwanted disturbance.
Om t-tester eller Dunnetts test används finns det inget behov av ett ANOVA-signifikans F-test för att gå vidare.
Recommended fish species are given in Appendix 2.
För närmare detaljer, se flödesschemat i tillägg 8.
The procedures for inclusion of new species are given in paragraph 2.
Resultaten bör rapporteras i tabeller som medelvärden av koncentrationen ± standardavvikelser för hanar, honor, intersexuella och odifferentierade var för sig.
Holding of parental fish
Den statistiska signifikansen för fenotypiska honor och fenotypiska hanar bör framhävas.
Details on holding the parental fish under satisfactory conditions may be found in OECD TG 210(1).
Faktiska koncentrationer av testkemikalien
Parental fish should be fed once or twice a day with appropriate food.
De faktiska koncentrationerna av testkemikalien i behållaren bör analyseras i frekvenser som beskrivs i punkt 34.
Handling of embryos and larvae
Man bör rapportera resultaten i tabeller som medelvärdet av koncentrationen ± standardavvikelser på replikat- och koncentrationsbasis med information om antalet prover, och då markera avvikelser från den genomsnittliga behandlingskoncentrationen ± 20 %.
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.
Exempel finns att tillgå i valideringsrapporten för testet på fiskars könsutveckling fas 2 (42).
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.
Resultaten bör tolkas med försiktighet när de uppmätta koncentrationerna av testkemikalien i testlösningen ligger på nivåer nära analysmetodens detektionsgräns.
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.
nominella testkoncentrationer, de uppmätta värdenas medelvärden och deras standardavvikelser i testkärlen samt den metod med vilken dessa erhölls (den analysmetod som används bör presenteras i en bilaga) och bevis för att mätningarna hänför sig till testkemikaliens koncentrationer i en äkta lösning, vattenkvaliteten i testbehållarna:
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.
pH, hårdhet, temperatur och halten löst syre,
The timing of this transfer varies with the species and transfer may not always be necessary.
belägg för att kontrollerna uppfyller giltighetskriterierna:
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.
Avvikelser från kriterierna för godkännande (i kontrollerna) bör lyftas fram.
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.
Resultaten bör presenteras som medelvärden ± standardavvikelse eller standardfel.
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.
Statistiken ska rapporteras åtminstone som LOEC och NOEC samt konfidensintervall.
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.
Man bör följa det statistiska flödesschemat (tillägg 8).
Details about chemical analysis and water collection can be found in paragraph 34.
Sumpter, J. P. och S. Jobling, 1995, ’Vitellogenesis As A Biomarker for Estrogenic Contamination of the Aquatic Environment’, Environmental Health Perspectives 103, s.
Flow-through system should be used if practically possible.
173–178.
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.
Andersen, L., P. Bjerregaard och B. Korsgaard ( 2003), ’Vitellogenin induction and brain aromatase activity in adult male and female zebrafish exposed to endocrine disrupters’, Fish Physiology and Biochemistry 28, s. 319–321.
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.
Hatano, H. Mizukami, M. Yamashita, R. Sakakibara och E. Tamiya (2002), ’Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin’, Comparative Biochemistry and Physiology C – Toxicology & Pharmacology 132, s.
A flow rate equivalent to at least five test chamber volumes per 24 hours has been found suitable (1).
161–169.
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.
Tatarazako, N., M. Koshio, H. Hori, M. Morita och T. Iguchi (2004), ’Validation of an enzyme-linked immunosorbent assay method for vitellogenin in the medaka’, Journal of Health Science 50, s.
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).
301–308.
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).
Eidem, J. K., H. Kleivdal, K. Kroll, N. Denslow, R. van Aerle, C. Tyler, G. Panter, T. Hutchinson och A. Goksoyr (2006), ’Development and validation of a direct homologous quantitative sandwich ELISA for fathead minnow (Pimephales promelas) vitellogenin.’
The use of solvents should be avoided but may be necessary in some cases in order to produce a suitably concentrated stock solution.
Aquatic Toxicology, 78, s.
Examples of suitable solvents are given in (36).
202–206.
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).
Holbech, H., Petersen, G. I., Norman, A., Örn, S, Norrgren, L. och Bjerregaard, P (2001b), ’Suitability of zebrafish as test organism for detection of endocrine disrupting chemicals.
For the semi-static technique, two different renewal procedures may be followed.
Comparison of vitellogenin in plasma and whole body homogenate from zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss)’, Nordiska ministerrådet, TemaNord 2001:597, s.
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.
48–51.
Conditions of Exposure
Orn, S., S. Yamani och L. Norrgren (2006), ’Comparison of vitellogenin induction, sex ratio, and gonad morphology between zebrafish and Japanese medaka after exposure to 17 alpha-ethinylestradiol and 17 beta-trenbolone’, Archives of Environmental Contamination and Toxicology 51, s.
Collection of eggs and duration
237–243.
To avoid genetic bias, eggs are collected from a minimum of three breeding pairs or groups, mixed and randomly selected to initiate the test.
Scholz, S. och N. Kluver (2009), ’Effects of Endocrine Disrupters on Sexual, Gonadal Development in Fish’, Sexual Development 3, s.
For the three-spined stickleback, see the description of artificial fertilisation in Appendix 11.
136–151.
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.
Fenske, M., G. Maack, C. Schafers och H. Segner (2005), ’An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio’, Environmental Toxicology and Chemistry 24, s.
The test should continue until sexual differentiation in the control group is completed (60 dph for Japanese medaka, the three-spined stickleback and zebrafish).
1088–1098.
Loading
Nagahama (2004), ’Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes’, Developmental Dynamics 231, s.
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).
518–526.
The eggs should be randomly distributed (by using statistical tables for randomisation) among treatments.
Shinomiya, A., H. Otake, K. Togashi, S. Hamaguchi och M. Sakaizumi (2004), ’Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations’, Zoological Science 21, s.
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.
613–619.
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.
Flick (2007), ’Collapse of a fish population after exposure to a synthetic estrogen’, Proceedings of the National Academy of Sciences of the United States of America 104, s.
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.
8897–8901.
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.
P., R. E. Evans, K. G. Wautier, K. H. Mills, P. J. Blanchfield, B. J. Park, C. L. Baron och K. A. Kidd (2009), ’Interspecies differences in biochemical, histopathological, and population responses in four wild fish species exposed to ethynylestradiol added to a whole lake’, Canadian Journal of Fisheries and Aquatic Sciences 66, s.
The photoperiod and water temperature should be appropriate for the test species (see Appendix 2 for experimental conditions for the FSDT).
1920–1935.
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.
Morthorst, J. E., H. Holbech och P. Bjerregaard (2010), ’Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations’, Aquatic Toxicology 98, s.
Surplus food and faeces should be removed, as necessary, to avoid accumulation of waste.
336–343.
As experience is gained, food and feeding regimes are continuously being refined to improve survival and optimise growth.
Kapitel C.14 i denna bilaga, Tillväxttest på unga exemplar av fisk.
Effort should therefore be made to confirm the proposed regime with acknowledged experts.
Kapitel C.4 i denna bilaga, Lätt bionedbrytbarhet.
Feeding should be withheld 24 hours before ending the test.
Kapitel C.37 i denna bilaga, 21-dagars test på fisk:
Examples of appropriate food items are listed in Appendix 2 (see also the OECD Fish Testing Framework (39).
En kortsiktig screening för ämnen med östrogen och androgen aktivitet samt aromatashämmande verkan.
Test chemicals should be spaced as described in Appendix 4.
Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., Segner, H (2007), ’Concentration- and time-dependent effects of the synthetic estrogen, 17 alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio’, Journal of Toxicology and Environmental Health – Del A, 70, 9–10, s.
A minimum of three test concentrations in at least four replicates should be used.
768–779.
The curve relating LC50 to period of exposure in the acute studies available should be considered when selecting the range of test concentrations.
Förkortningar och definitioner
The maximum test concentration should be 10 % of the LC50 on the larval/juvenile life-stage.
luftmättnadsvärdet
Controls
Biomarkör
A dilution water control (≥ 4 replicates) and, if relevant, a solvent control (≥ 4 replicates) should be run in addition to the test concentrations.
orsakar effekter på individnivå
Only solvents that have been investigated not to have any statistical significant influence on the test endpoints should be used in the test.
ett ämne eller en blandning
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.
dagar efter kläckningen
However, every effort should be made to avoid the use of such solvent or keep solvent's concentrations to a minimum.
Y-länkad DM-domängen som krävs för den hanliga utvecklingen i risfiskar
Frequency of Analytical Determinations and Measurements
Fiskvikt
Chemical analysis of the test chemical concentration should be performed before initiation of the test to check compliance with the acceptance criteria.
fiskens våtvikt (avtorkad)
All replicates should be analysed individually at the beginning and termination of the test.
Test på fiskars könsutveckling
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…).
stressaxeln, bestående av hypotalamus, hypofysen och binjurebarken
If samples are stored to be analysed at a later time, the storage method of the samples should be previously validated.
Intersexuella fiskar
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.
fiskar med mer än en oocyt i testikeln per sex analyserade preparat eller spermatogena celler i äggstockarna (ja/nej)
During the test, dissolved oxygen, pH, total hardness, conductivity, salinity (if relevant), and temperature should be measured in all test chambers.
våtvikten fisk per vattenvolym
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.
verkningssätt
Temperature should preferably be monitored continuously in at least one test chamber.
PCR med omvänd transkription
Results should be based on measured concentrations.
Odifferentierad fisk
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.
fisk med könskörtlar som inte har några märkbara könsceller
Observations and measurements
Försöksbetingelser för testet på fiskars könsutveckling (sötvattensarter)
Stage of embryonic development
Japansk risfisk (Oryzias latipes)
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.
Sebrafisk (Danio rerio)
Hatching and survival
Storspigg (Gasterostreus aculeatus)
for eggs:
12–16 timmar ljus, 8–12 timmar mörker
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;
Behållarens minsta storlek
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.
Testorganismernas ålder i början av exponeringen
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.
Antal ägg per testkärl
These observations will result in some or all of the following data being available for reporting:
Rent yt- eller brunnsvatten eller rekonstituerat vatten
cumulative mortality;
Den kemiska exponeringens varaktighet
numbers of healthy fish at end of test;
Kläckningsframgång, överlevnad, makromorfologi, VTG könskörtlarnas histologi, genetiskt kön könsfördelning
length and weight of surviving animals;
Testets godtagbarhetskriterier för sammanlagda kontrollreplikat
Sampling of fish
Tillväxt (den avtorkade fiskens våtvikt) > 150 mg
For the VTG measurements, a sub-sampling of at least 16 fish from each replicate is accepted.
Könsfördelning (% hanar och honor) 30–70 %
The sampling procedure for VTG and sex determination is dependent on the VTG analysis method:
KONCENTRATION
Head/tail homogenate method for VTG analysis
Partiklar
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).
Kolumn (antal koncentrationer mellan 100 och 10 eller mellan 10 och 1) [1]
The body part of the fish is numbered and fixed in an appropriate fixative for histological evaluation (22).
Mittpunkterna mellan koncentrationerna i kolumn (x) finns i kolumn (2x + 1).