natgillin/translations-raw huggingface dataset:
Genetic sex is examined via genetic markers when they exist in a given fish species.
Andersen, L., K. Kinnberg, H. Holbech, B. Korsgaard a P. Bjerregaard (2004), ‚Evaluation of a 40 day assay for testing endocrine disrupters: Effects of an anti-estrogen and an aromatase inhibitor on sex ratio and vitellogenin concentrations in juvenile zebrafish (Danio rerio)‘, Fish Physiology and Biochemistry 30, s.
In Japanese medaka the female XX or male XY genes can be detected by Polymerase Chain-Reaction (PCR), or the Y-linked DM domain gene (DMY) can be analysed (DMY negative or positive) as described in (23) (24).
257 – 266.
In three-spined stickleback, there is an equivalent PCR method for genetic sex determination described in Appendix 10.
Morthorst, J.E., H. Holbech a P. Bjerregaard (2010), ‚Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations‘, Aquatic Toxicology 98, s.
Where the genetic sex can be individually linked to the phenotypic sex, the power of the test is improved and therefore genetic sex should be determined in species with documented genetic sex markers.
336 – 343.
The two core endocrine endpoints, VTG and sex ratio, can in combination demonstrate the endocrine mode of action (MOA) of the chemical (Table 1).
Metcalf (2003), ‚Effects of the antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes)‘, Aquatic Toxicology 63, s.
The sex ratio is a population relevant biomarker (25) (26) and for some well defined modes of action, the FSDT results may be used for hazard and risk assessment purposes when deemed appropriate by the regulatory agency.
391 – 403.
These modes of action are at present oestrogens, androgens and steroidogenesis inhibitors.
Tyler (2004), ‚Successful detection of (anti-) androgenic and aromatase inhibitors in pre-spawning adult fathead minnows (Pimephales promelas) using easily measured endpoints of sexual development‘, Aquatic Toxicology 70, s.
Reaction of the endocrine endpoints to different modes of action of chemicals
11 – 21.
↑ = increasing, ↓ = decreasing, — = not investigated
Petersen a P. Bjerregaard (2007), ‚Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio)‘, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 145, s.
References
165 – 170.
Weak oestrogen agonist
Kapitola C.14 tejto prílohy, rastová skúška na mladých jedincoch.
↑♀ or ↑Undiff
Kapitola C.4 tejto prílohy, ľahká biodegradovateľnosť.
Strong oestrogen agonist
Kapitola C.37 tejto prílohy, 21-Day Fish Assay:
↑♀ or ↑Undiff, No ♂
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-Part A, 70, 9-10 s 768 – 779.
Oestrogen antagonist
Skratky a vymedzenie pojmov
↓♀, ↑Undiff.
Vrcholový sledovaný parameter
Androgen agonist
účinok pôsobiaci na úrovni populácie.
↑♂, No ♀
rozpustnosť vzdušného kyslíka.
Androgen antagonist
účinok pôsobiaci na úrovni jedinca.
↑Intersex
látka alebo zmes.
Aromatase inhibitor
dni po vyliahnutí.
The FSDT does not cover the reproductive life stage of the fish and therefore chemicals that are suspected to affect reproduction at lower concentrations than sexual development should be examined in a test that covers reproduction.
gén domény DM špecifický pre chromozómom Y potrebný na vývin samčekov rýb rodu medaka.
Definitions for the purpose of this Test Method are given in Appendix 1.
enzýmové imunosorbentové stanovenie
The in vivo FSDT is intended to detect chemicals with androgenic and oestrogenic properties as well as anti-androgenic, anti-oestrogenic and steroidogenesis inhibiting properties.
Hmotnosť ryby
The FSDT validation phases (1 and 2) did cover oestrogenic, androgenic and steroidogenesis inhibiting chemicals.
mokrá hmotnosť ryby (vytretej do sucha).
The effects in the FSDT of oestrogen- and androgen antagonists can be seen in Table 1 but these MOA are less documented at present time.
test pohlavného vývinu rýb.
In the test, fish are exposed, from newly fertilised egg until the completion of sexual differentiation, to at least three concentrations of the test chemical dissolved in water.
os hypotalamus – hypofýza – gonády.
The test conditions should be flow-through unless not possible due to the availability or nature (e.g. limited solubility) of the test chemical.
Intersexuálna ryba
The test starts with the placing of newly fertilised eggs (before cleavage of the blastodisc) in the test chambers.
ryba s viac ako jedným oocytom v semenníkoch na 6 analyzovaných rezov alebo so spermatogénnymi bunkami (áno/nie) vo vaječníkoch.
The loading of the chambers is described for each species in paragraph 27.
mokrá hmotnosť rýb na objem vody.
For the validated fish species, Japanese medaka, the three-spined stickleback and zebrafish, the test is terminated at 60 dph.
spôsob pôsobenia.
At test termination, all fish are euthanised humanely.
polymerázová reťazová reakcia s reverznou transkriptázou.
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;
Nediferencovaná ryba
optionally, histopathology (e.g. staging of gonads, severity of intersex) can be performed.
ryba s gonádami bez rozoznateľných zárodočných buniek.
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).
vitellogenín.
An overview of relevant test conditions specific for validated species:
Experimentálne podmienky testu FSDT (sladkovodné druhy)
Japanese medaka, the three-spined stickleback and zebrafish is provided in Appendix 2.
Medaka japonská (Oryzias latipes)
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.
Dánio pruhované (Danio rerio)
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.
Pichľavka sivá (Gasterostreus aculeatus)
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).
Prietokový alebo semistatický
Test acceptance criteria
Žiarivky (široké spektrum)
The dissolved oxygen concentration should be at least 60 per cent of the air saturation value (ASV) throughout the test;
Intenzita svetla:
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);
10 – 20 μE/m2/s, 540 – 1080 luxov alebo 50 – 100 ft-c (úrovne v laboratórnom prostredí)
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;
12 – 16 hod. svetlo, 8 – 12 hod. tma
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;
Minimálna veľkosť komory
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):
Jednotlivé komory by mali obsahovať minimálne 7 l vody
Japanese medaka
Vek testovacích organizmov na začiatku expozície
Three-spined stickleback
Čerstvo oplodnené vajíčka (skoré štádium blastuly)
Growth
Čerstvo oplodnené vajíčka
Fish wet weight, blotted dry
Počet vajíčok na testovanú koncentráciu
> 75 mg
Minimálne 120
Length (standard length)
Minimálne tri (plus primerané kontrolné skupiny)
> 20 mm
Počet replikátov na testovanú koncentráciu
> 14 mm
Minimálne štyri (okrem kontrolných skupín, kde sa počet určí podľa funkcie druhej odmocniny)
Sex ratio (% males or females)
Živé žiabronôžky rodu Artemia, dospelé zmrazené žiabronôžky soľné, vločkové krmivo atď. Odporúča sa kŕmiť dvakrát denne.
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.
Špeciálna potrava pre mladé ryby, živé žiabronôžky rodu Artemia, dospelé zmrazené žiabronôžky soľné, vločkové krmivo atď. Odporúča sa kŕmiť dvakrát denne.
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.
Žiadne, pokiaľ koncentrácia rozpusteného kyslíka neklesne pod 60 % saturáciu
Test chambers
Žiadne, pokiaľ koncentrácia rozpusteného kyslíka neklesne pod 70 % saturáciu
Any glass, stainless steel or other chemically inert chambers can be used.
Čistá povrchová, studňová alebo rekonštituovaná voda
The dimensions of the chambers should be large enough to allow compliance with loading rate criteria given below.
Trvanie expozície testovanej chemikálii
It is desirable that test chambers be randomly positioned in the test area.
Úspešné vyliahnutie, prežitie, výrazná morfológia, VTG, histológia gonád, genetické pohlavie, pomer pohlaví
A randomised block design with each concentration being present in each block is preferable to a completely randomised design.
Úspešné vyliahnutie, prežitie, výrazná morfológia, VTG, histológia gonád, pomer pohlaví
The test chambers should be shielded from unwanted disturbance.
Kritériá prijateľnosti testu pre spojené replikáty kontrolných skupín
Recommended fish species are given in Appendix 2.
Úspešné vyliahnutie > 80 %
The procedures for inclusion of new species are given in paragraph 2.
Prežitie po vyliahnutí ≥ 70 %
Holding of parental fish
Rast (mokrá hmotnosť ryby vytretej do sucha) > 150 mg
Details on holding the parental fish under satisfactory conditions may be found in OECD TG 210(1).
Rast (mokrá hmotnosť ryby vytretej do sucha) > 75 mg
Parental fish should be fed once or twice a day with appropriate food.
Rast (mokrá hmotnosť ryby vytretej do sucha) > 120 mg
Handling of embryos and larvae
Dĺžka (štandardná dĺžka) > 20 mm
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.
Dĺžka (štandardná dĺžka) > 14 mm
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.
Dĺžka (štandardná dĺžka) > 20 mm
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.
Pomer pohlaví (% samčekov alebo samičiek) 30 – 70 %
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.
Chemické charakteristiky prijateľnej riediacej vody
The timing of this transfer varies with the species and transfer may not always be necessary.
KONCENTRÁCIA
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.
Tuhé častice
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.
Podľa testovacej metódy C.14/Usmernenie o testovaných koncentráciách
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.
Stĺpec (počet koncentrácií medzi 100 a 10 alebo medzi 10 a 1) [1]
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.
Zo stĺpca možno vybrať sériu troch (alebo viacerých) za sebou idúcich koncentrácií.
Details about chemical analysis and water collection can be found in paragraph 34.
Stredné body medzi koncentráciami v stĺpci (x) sa nachádzajú v stĺpci (2x + 1).
Flow-through system should be used if practically possible.
Uvedené hodnoty môžu predstavovať koncentrácie vyjadrené ako percento na objem alebo hmotnosť (mg/l alebo μg/l).
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.
Hodnoty je podľa potreby možné násobiť alebo deliť akoukoľvek mocninou 10.
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.
Hodnoty v stĺpci 1 sa môžu použiť v prípade značnej neistoty v súvislosti s úrovňou toxicity.
A flow rate equivalent to at least five test chamber volumes per 24 hours has been found suitable (1).
Usmernenie na homogenizáciu hlavy a chvosta z mladých jedincov dánia pruhovaného, čereble potočnej, pichľavky sivej a medaky japonskej
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.
Účelom tohto oddielu je opísať postupy, ktoré sa vykonávajú pred kvantifikáciou koncentrácie VTG.
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).
Možno použiť aj iné postupy, ktoré vedú k porovnateľnej kvantifikácii VTG.
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).
Namiesto homogenátu hlavy/chvosta je možné určiť koncentráciu VTG aj v krvnej plazme alebo pečeni.
The use of solvents should be avoided but may be necessary in some cases in order to produce a suitably concentrated stock solution.
Oddelí sa hlava a chvost ryby v súlade s opisom testu.
Examples of suitable solvents are given in (36).
Všetky nástroje a doska na rezanie sa musia medzi manipuláciami s jednotlivými rybami riadne vyčistiť a opláchnuť (napr. 96 % etanolom), aby sa zabránilo ‚znečisteniu VTG‘ zo samičiek alebo indukovaných samčekov na neindukovaných samčekov.
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).
Po vytvorení homogénnej hmoty sa pridá množstvo ľadovo studeného homogenizačného tlmivého roztoku zodpovedajúce štvor- až desaťnásobku hmotnosti tkaniva (poznamenajte si riedenie).
For the semi-static technique, two different renewal procedures may be followed.
Vzorky sa umiestnia na ľad až do odstredenia pri teplote 4 °C pri 50000 g počas 30 minút.
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.
Pipetou preneste dávky 20 – 50 μl (poznamenajte si množstvo) supernatantu aspoň do dvoch skúmaviek tak, že namočíte hrot pipety pod vrstvu tuku na povrchu a starostlivo odsajete supernatant bez tukových alebo peletových frakcií.
Conditions of Exposure
Počas používania ho umiestnite na ľad.
Collection of eggs and duration
Homogenizačný tlmivý roztok:50 mM Tris-HCl, pH 7,4;
To avoid genetic bias, eggs are collected from a minimum of three breeding pairs or groups, mixed and randomly selected to initiate the test.
1 % koktail inhibítorov proteázy (Sigma):
For the three-spined stickleback, see the description of artificial fertilisation in Appendix 11.
12 ml Tris-HCl, pH 7,4 + 120 μl koktailu inhibítorov proteázy (alebo ekvivalentného koktailu inhibítorov proteázy).TRIS:
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.
TRIS-ULTRA PURE (ICN)Koktail inhibítorov proteázy:
The test should continue until sexual differentiation in the control group is completed (60 dph for Japanese medaka, the three-spined stickleback and zebrafish).
Od firmy Sigma (pre tkanivo cicavcov), číslo produktu P 8340.
Loading
Usmernenie na kvantifikáciu vitellogenínu z homogenátu hlavy a chvosta dánia pruhovaného (danio rerio) (upravené z holbecha a kol., 2001). možno použiť aj iné postupy využívajúce homológne protilátky a štandardy.
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).
Mikrotitračné platne (certifikované Maxisorp F96, Nunc, Roskilde, Dánsko) potiahnuté 5 μg/ml IgG proti lipovitellínu dánia pruhovaného sa rozmrazia a trikrát premyjú premývacím tlmivým roztokom (*).
The eggs should be randomly distributed (by using statistical tables for randomisation) among treatments.
Purifikovaný štandardný roztok vitellogenínu dánia pruhovaného [1] sa sériovo zriedi na 0,2; 0,5; 1; 2; 5;
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.
10 a 20 ng/ml v riediacom tlmivom roztoku (**).
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.
Vzorky sa v riediacom tlmivom roztoku riedia aspoň 200-krát (aby nedošlo k efektu matrice) a aplikujú sa na platne.
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.
Kontrola skúšky sa aplikuje ako duplikát.
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.
Do každej jamky sa aplikuje 150 μl.
The photoperiod and water temperature should be appropriate for the test species (see Appendix 2 for experimental conditions for the FSDT).
Štandardy sa aplikujú v duplikátoch a vzorky v triplikátoch.
Food and feeding are critical, and it is essential that the correct food for each stage is supplied at appropriate time intervals and at a level sufficient to support normal growth.
Inkubujte cez noc pri teplote 4 °C na trepačke.
Feeding should be ad libitum whilst minimising the surplus.
Platne sa päťkrát premyjú premývacím tlmivým roztokom. (*)
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.
HRP pripojená k dextranovému reťazcu (napr. AMDEX A/S, Dánsko) a konjugované protilátky sa zriedia v premývacom tlmivom roztoku.
Surplus food and faeces should be removed, as necessary, to avoid accumulation of waste.
Skutočné riedenie sa líši podľa šarže a veku.
As experience is gained, food and feeding regimes are continuously being refined to improve survival and optimise growth.
Do každej jamky sa aplikuje 150 μl a platne sa 1 hodinu inkubujú pri izbovej teplote na trepačke.
Effort should therefore be made to confirm the proposed regime with acknowledged experts.
Platne sa päťkrát premyjú premývacím tlmivým roztokom (*) a spodok platní sa opatrne vyčistí etanolom.
Feeding should be withheld 24 hours before ending the test.
Do každej jamky sa aplikuje 150 μl roztoku TMB plus (***).
Examples of appropriate food items are listed in Appendix 2 (see also the OECD Fish Testing Framework (39).
Platňu chráňte pred svetlom staniolom a sledujte vývoj farby na trepačke.
Test chemicals should be spaced as described in Appendix 4.
Keď sa plne vytvorí krivka štandardného roztoku, enzymatická aktivita sa zastaví pridaním 150 μl 0,2 M H2SO4 do každej jamky.
A minimum of three test concentrations in at least four replicates should be used.
Premývací tlmivý roztok:
The curve relating LC50 to period of exposure in the acute studies available should be considered when selecting the range of test concentrations.
Upravte pH na 7,3 a doplňte na 5 l milipórovej H2O.
Five test concentrations are recommended if the data are to be used for risk assessment.
Skladujte pri teplote 4 °C.
Concentrations of the chemical higher than 10 % of the acute adult LC50 or 10 mg/l, whichever is the lower, need not be tested.
Riediaci tlmivý roztok:
The maximum test concentration should be 10 % of the LC50 on the larval/juvenile life-stage.
PBS-Stock****
Controls
Upravte pH na 7,3 a doplňte na 1 l milipórovej H2O.
A dilution water control (≥ 4 replicates) and, if relevant, a solvent control (≥ 4 replicates) should be run in addition to the test concentrations.
TMB plus je ‚hotový‘ substrát vyrobený spoločnosťou KemEnTec (Dánsko).
Only solvents that have been investigated not to have any statistical significant influence on the test endpoints should be used in the test.
Je citlivý na svetlo.
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.
HPO4.2H2O
However, every effort should be made to avoid the use of such solvent or keep solvent's concentrations to a minimum.
Upravte pH na 6,8 a doplňte na 2 l milipórovou vodou.
Frequency of Analytical Determinations and Measurements
Skladujte pri izbovej teplote.
Chemical analysis of the test chemical concentration should be performed before initiation of the test to check compliance with the acceptance criteria.
Battelle AP4.6.04 (1,18 mg/ml (AAA)), purifikované podľa:
All replicates should be analysed individually at the beginning and termination of the test.
Usmernenie na prípravu rezov tkaniva na účely určenia pohlavia a štádia gonád
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…).
Účelom tohto oddielu je opísať postupy, ktoré sa vykonávajú pred hodnotením histologických rezov.
If samples are stored to be analysed at a later time, the storage method of the samples should be previously validated.
Možno použiť aj iné postupy, ktoré vedú k podobnému určeniu pohlavia a štádia gonád.
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.
Až na niekoľko výnimiek sú tieto postupy podobné pre medaku japonskú (JMD) a dánio pruhované (ZF).
During the test, dissolved oxygen, pH, total hardness, conductivity, salinity (if relevant), and temperature should be measured in all test chambers.
Usmrtenie, pitva a fixácia tkaniva
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.
Ciele:
Temperature should preferably be monitored continuously in at least one test chamber.
Umožniť humánne usmrtenie rýb.
Results should be based on measured concentrations.
Získať potrebné telesné miery a hmotnosť.