natgillin/translations-raw huggingface dataset:
This is the case for zebrafish, three-spined stickleback and Japanese medaka and also the partially validated species fathead minnow;
Poglavje C.14 te priloge, Preskus rasti ribjih mladic.
species-specific Enzyme-Linked Immunosorbent Assay (ELISA) methods using immunochemistry for the quantification of VTG are available (5) (10) (11) (12) (13) (14) (15) (16).
Poglavje C.4 te priloge, Razgradnja – biokemijska potreba po kisiku.
In Japanese medaka and zebrafish, there is a good correlation between VTG measured from blood plasma, liver and homogenate samples although homogenates tend to show slightly lower values than plasma (17) (18) (19).
Poglavje C.37 te priloge, 21-dnevni preskus rib:
Appendix 5 provides the recommended procedures for sample collection for VTG analysis.
kratkotrajno presejalno preskušanje estrogenega in androgenega delovanja ter zaviranja aromataze.
Change in the phenotypic sex ratio (proportions of sex) is an endpoint reflecting sex reversal.
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 str. 768–779.
In principle, oestrogens, anti-oestrogens, androgens, anti-androgens and steroidogenesis inhibiting chemicals can affect the sex ratio of developing fish (20).
Okrajšave in opredelitev pojmov
It has been shown that this sex reversal is partly reversible in zebrafish (21) following oestrogen-like chemical exposure, whereas sex reversal following androgen-like chemical exposure is permanent (30).
Zgornja končna točka
The sex is defined as female, male, intersex (both oocytes and spermatogenetic cells in one gonad) or undifferentiated, determined in individual fish via histological examination of the gonads.
povzroča učinke na ravni populacije.
Guidance is given in Appendix 7 and in the OECD Guidance Document on the Diagnosis of Endocrine-Related Histopathology of Fish Gonads (22).
nasičenost z zrakom (air saturation value).
Genetic sex is examined via genetic markers when they exist in a given fish species.
povzroča učinke na ravni posameznega osebka.
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).
snov ali zmes.
In three-spined stickleback, there is an equivalent PCR method for genetic sex determination described in Appendix 10.
dnevi po izvalitvi (days post hatch).
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.
na kromosom Y vezan gen z domeno DM, ki je potreben za razvoj samcev medak.
The two core endocrine endpoints, VTG and sex ratio, can in combination demonstrate the endocrine mode of action (MOA) of the chemical (Table 1).
encimski imunski test.
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.
Teža ribe
These modes of action are at present oestrogens, androgens and steroidogenesis inhibitors.
mokra teža ribe (osušene s pivnikom).
Reaction of the endocrine endpoints to different modes of action of chemicals
preskus spolnega razvoja rib.
↑ = increasing, ↓ = decreasing, — = not investigated
os hipotalamus – hipofiza – spolne žleze.
References
Dvospolne ribe
Weak oestrogen agonist
ribe z več kot enim oocitom v testisu na 6 analiziranih delov ali spermatogenimi celicami v jajčnikih (da/ne).
↑♀ or ↑Undiff
mokra teža rib na prostornino vode.
Strong oestrogen agonist
način delovanja.
↑♀ or ↑Undiff, No ♂
verižna reakcija s polimerazo z reverzno transkriptazo.
Oestrogen antagonist
Nediferencirane ribe
↓♀, ↑Undiff.
ribe s spolnimi žlezami, ki ne kažejo zaznavnih zarodnih celic.
Androgen agonist
vitelogenin.
↑♂, No ♀
Poskusni pogoji za preskus spolnega razvoja rib (sladkovodne vrste)
Androgen antagonist
japonska medaka (Oryzias latipes)
↑Intersex
cebrica (Danio rerio)
Aromatase inhibitor
zet (Gasterostreus aculeatus)
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.
pretočni ali polstatični
Definitions for the purpose of this Test Method are given in Appendix 1.
fluorescenčne sijalke (široki spekter)
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.
Jakost svetlobe
The FSDT validation phases (1 and 2) did cover oestrogenic, androgenic and steroidogenesis inhibiting chemicals.
10–20 μE/m2/s, 540–1080 luksov ali 50–100 ft-c (ravni v laboratoriju)
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.
12–16 ur svetlobe, 8–12 ur teme
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.
Najmanjša velikost komore
The test conditions should be flow-through unless not possible due to the availability or nature (e.g. limited solubility) of the test chemical.
posamezne komore morajo vsebovati vsaj 7 l vode
The test starts with the placing of newly fertilised eggs (before cleavage of the blastodisc) in the test chambers.
Starost preskusnih organizmov ob začetku izpostavljenosti
The loading of the chambers is described for each species in paragraph 27.
novo oplojene ikre (zgodnji stadij blastule)
For the validated fish species, Japanese medaka, the three-spined stickleback and zebrafish, the test is terminated at 60 dph.
novo oplojene ikre
At test termination, all fish are euthanised humanely.
Št. iker na tretiranje
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;
najmanj 120
optionally, histopathology (e.g. staging of gonads, severity of intersex) can be performed.
najmanj 3 (plus ustrezno št. kontrol)
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).
Št. ponovitev na tretiranje
An overview of relevant test conditions specific for validated species:
najmanj 4 (razen v primeru razporeditve v kontrole na podlagi kvadratnega korena)
Japanese medaka, the three-spined stickleback and zebrafish is provided in Appendix 2.
živa Artemia, zamrznjeni odrasli morski rakci, hrana v kosmičih itd.; priporočeno je hranjenje dvakrat na dan
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.
posebna hrana za zarod, živa Artemia, zamrznjeni odrasli morski rakci, hrana v kosmičih itd.; priporočeno je hranjenje dvakrat na dan
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.
brez, razen če koncentracija raztopljenega kisika pade pod 60 % nasičenosti
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).
brez, razen če koncentracija raztopljenega kisika pade pod 70 % nasičenosti
Test acceptance criteria
čista površinska voda, vodovodna voda ali modelna razredčevalna voda
The dissolved oxygen concentration should be at least 60 per cent of the air saturation value (ASV) throughout the test;
Trajanje izpostavljenosti preskusni kemikaliji
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);
uspešnost izvalitve, preživetje, morfologija velikih organov, VTG histologija spolnih žlez, genetski spol razmerje med spoloma
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;
uspešnost izvalitve, preživetje morfologija velikih organov, VTG histologija spolnih žlez, razmerje med spoloma
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;
Merila sprejemljivosti preskusa za združene ponovitve kontrol
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):
uspešna izvalitev > 80 %
Japanese medaka
preživetje po izvalitvi ≥ 70 %
Three-spined stickleback
rast (mokra teža ribe, osušena s pivnikom) > 150 mg
Growth
rast (mokra teža ribe, osušena s pivnikom) > 75 mg
Fish wet weight, blotted dry
rast (mokra teža ribe, osušena s pivnikom) > 120 mg
> 75 mg
dolžina (standardna dolžina) > 20 mm
Length (standard length)
dolžina (standardna dolžina) > 14 mm
> 20 mm
dolžina (standardna dolžina) > 20 mm
> 14 mm
razmerje med spoloma (% samcev ali samic) 30 %–70 %
Sex ratio (% males or females)
Kemijske značilnosti sprejmljive vode za redčenje
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.
KONCENTRACIJA
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.
delci
Test chambers
Iz preskusne metode C.14/Smernica za preskusne koncentracije
Any glass, stainless steel or other chemically inert chambers can be used.
Stolpec (število koncentracij med 100 in 10 ali med 10 in 1) [1]
The dimensions of the chambers should be large enough to allow compliance with loading rate criteria given below.
Iz stolpca se lahko izbere serija treh (ali več) zaporednih koncentracij.
It is desirable that test chambers be randomly positioned in the test area.
Vmesne vrednosti med koncentracijami v stolpcu (x) so v stolpcu (2x + 1).
A randomised block design with each concentration being present in each block is preferable to a completely randomised design.
Navedene vrednosti lahko predstavljajo koncentracije, izražene v odstotkih na prostornino ali težo (mg/l ali μg/l).
The test chambers should be shielded from unwanted disturbance.
Vrednosti se lahko množijo ali delijo s katero koli potenco števila 10, kot je ustrezno.
Recommended fish species are given in Appendix 2.
Stolpec 1 se lahko uporabi, če je ugotovljena znatna negotovost stopnje strupenosti.
The procedures for inclusion of new species are given in paragraph 2.
Smernica za homogenizacijo glave in repa mladic cebrice, črnoglavega pisanca, zeta in japonske medake
Holding of parental fish
Namen tega razdelka je opisati postopke, ki se izvedejo pred kvantifikacijo koncentracije VTG.
Details on holding the parental fish under satisfactory conditions may be found in OECD TG 210(1).
Uporabijo se lahko tudi drugi postopki, katerih rezultat je primerljiva kvantifikacija VTG.
Parental fish should be fed once or twice a day with appropriate food.
Koncentracija VTG se lahko namesto v homogenatu glave/repa določi v krvni plazmi ali jetrih.
Handling of embryos and larvae
Glava in rep ribe se odrežeta v skladu z opisom preskusa.
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.
med ravnanji s posameznimi ribami je treba vse instrumente za seciranje in rezalno desko sprati in ustrezno očistiti (npr. s 96-odstotnim etanolom), da se prepreči ‚onesnaženje‘ neinduciranih samcev z VTG samic ali induciranih samcev.
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.
Ko dobimo homogeno maso, se ji doda 4–10-kratna teža tkiva ledeno mrzlega homogenizacijskega pufra (paziti je treba na raztopino).
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.
Vzorci se postavijo na led do začetka centrifugiranja pri 4 °C in 50000 g za 30 minut.
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.
S pipeto se supernatant odmeri na dele po 20 do 50 μl (paziti je treba na količino) v vsaj dve epruveti, tako da se konica pipete potopi pod plast maščobe na površini in supernatant previdno izsesa brez delcev maščobe ali peletov.
The timing of this transfer varies with the species and transfer may not always be necessary.
Med uporabo ga je treba postaviti na led.
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.
Homogenizacijski pufer:50 mM Tris-HCl pH 7,4;
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.
dstotna zmes inhibitorja protaze (Sigma):
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.
12 ml Tris-HCl pH 7,4 + 120 μl zmesi inhibitorja protaze (ali enakovredne zmesi inhibitorja protaze).TRIS:
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.
TRIS ULTRA PURE (ICN)Zmes inhibitorja protaze:
Details about chemical analysis and water collection can be found in paragraph 34.
Sigma (za tkivo sesalcev), številka izdelka P 8340.
Flow-through system should be used if practically possible.
Smernica za kvantifikacijo vitelogenina v homogenatu glave in repa pri cebricah (Danio rerio) (spremenjen na podlagi Holbch idr., 2001). uporabijo se lahko drugi postopki, pri katerih se uporabljajo homologna antitelesa in raztopine
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.
Mikrotiterske plošče (potrjena Maxisorp F96, Nunc, Roskilde, Danska), predhodno premazane s 5 μg/ml lipovitelin-IgG proti cebricam, se odtajajo in trikrat sperejo s pufrom za spiranje (*).
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.
Prečiščena raztopina vitelogenina [1] pri cebricah se serijsko razredči na 0,2, 0,5, 1, 2, 5, 10 in 20 ng/ml v pufru za redčenje (**), vzorci pa se razredčijo vsaj 200-krat (da se prepreči vpliv matriksa) v pufru za redčenje in vnesejo na plošče.
A flow rate equivalent to at least five test chamber volumes per 24 hours has been found suitable (1).
Kontrola preskusa se ponovi dvakrat.
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.
V vsako vdolbinico se doda 150 μl.
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).
Raztopine se dodajo v ponovitev in vzorce v treh ponovitvah.
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).
Čez noč se na stresalniku inkubirajo pri 4 °C.
The use of solvents should be avoided but may be necessary in some cases in order to produce a suitably concentrated stock solution.
Plošče se petkrat sperejo s pufrom za spiranje (*).
Examples of suitable solvents are given in (36).
Hrenova peroksidaza (HRP), spojena z dekstranovo verigo (npr. AMDEX A/S, Danska), in konjugirana protitelesa se razredčijo v pufru za spiranje;
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).
dejanska raztopina se razlikuje po seriji in starosti.
For the semi-static technique, two different renewal procedures may be followed.
V vsako vdolbinico se doda 150 μl, plošče pa se eno uro inkubirajo na stresalniku pri sobni temperaturi.
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.
Plošče se petkrat sperejo s pufrom za spiranje (*), dno plošč pa se skrbno očisti z etanolom.
Conditions of Exposure
V vsako vdolbinico se doda 150 μl TMB plus (***).
Collection of eggs and duration
Ploščo je treba pred svetlobo zaščititi z aluminijasto folijo ter opazovati razvoj barv na stresalniku.
To avoid genetic bias, eggs are collected from a minimum of three breeding pairs or groups, mixed and randomly selected to initiate the test.
Ko je umeritvena krivulja popolnoma razvita, se aktivnost encimov ustavi z vnosom 150 μl 0,2 M H2SO4 v vsako vdolbinico.
For the three-spined stickleback, see the description of artificial fertilisation in Appendix 11.
Pufer za spiranje:
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.
založni PBS (****)
The test should continue until sexual differentiation in the control group is completed (60 dph for Japanese medaka, the three-spined stickleback and zebrafish).
Uravnati pH na 7,3 in napolniti do 5 l z Millipore H2O.
Loading
Hraniti pri 4 °C.
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).
Pufer za redčenje:
The eggs should be randomly distributed (by using statistical tables for randomisation) among treatments.
založni PBS (****)
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.
Uravnati pH na 7,3 in napolniti do 1 l z Millipore H2O.
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.
TMB plus je substrat, pripravljen za uporabo, ki ga proizvaja KemEnTec (Danska).
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.
Občutljiv je na svetlobo.
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.
Založni PBS
The photoperiod and water temperature should be appropriate for the test species (see Appendix 2 for experimental conditions for the FSDT).
HPO4 2H2O
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.
Battelle AP4.6.04 (1,18 mg/ml (AAA)), prečiščena v skladu z:
Surplus food and faeces should be removed, as necessary, to avoid accumulation of waste.
Smernica za pripravo tkivnih rezin za določanje spola in faze spolnih žlez
As experience is gained, food and feeding regimes are continuously being refined to improve survival and optimise growth.
Cilj tega razdelka je opisati postopke, ki se izvedejo pred oceno histoloških rezin.
Effort should therefore be made to confirm the proposed regime with acknowledged experts.
Uporabijo se lahko drugi postopki, katerih rezultat je podobna določitev spola in faze spolnih žlez.
Feeding should be withheld 24 hours before ending the test.
Ti postopki so z nekaj izjemami podobni za japonsko medako (JMD) in cebrico (ZF).
Examples of appropriate food items are listed in Appendix 2 (see also the OECD Fish Testing Framework (39).
Evtanazija, nekropsija in fiksacija tkiva
Test chemicals should be spaced as described in Appendix 4.
Cilji:
A minimum of three test concentrations in at least four replicates should be used.
zagotoviti humano žrtvovanje ribe;
The curve relating LC50 to period of exposure in the acute studies available should be considered when selecting the range of test concentrations.
pridobiti potrebne teže telesa in meritve;
Five test concentrations are recommended if the data are to be used for risk assessment.
oceniti sekundarne spolne značilnosti;
Concentrations of the chemical higher than 10 % of the acute adult LC50 or 10 mg/l, whichever is the lower, need not be tested.
secirati tkivo za analizo VTG;
The maximum test concentration should be 10 % of the LC50 on the larval/juvenile life-stage.
fiksirati spolne žleze.
Controls
Postopki:
A dilution water control (≥ 4 replicates) and, if relevant, a solvent control (≥ 4 replicates) should be run in addition to the test concentrations.
ribo je treba žrtvovati neposredno pred nekropsijo.
Only solvents that have been investigated not to have any statistical significant influence on the test endpoints should be used in the test.
Če ni na voljo več obducentov, se zato ne sme usmrtiti več rib hkrati;
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.
riba se z majhnim sakom odstrani iz preskusne komore in prenese na območje za nekropsijo v posodi za prenos;
However, every effort should be made to avoid the use of such solvent or keep solvent's concentrations to a minimum.
določi se mokra teža ribe;
Frequency of Analytical Determinations and Measurements
Za pripravo tkiv za analizo VTG se lahko riba postavi na plutovinasto ploščo na mizico stereomikroskopa;
Chemical analysis of the test chemical concentration should be performed before initiation of the test to check compliance with the acceptance criteria.
pri cebrici se glava odreže neposredno za prsno plavutjo, rep pa neposredno za hrbtno plavutjo;
All replicates should be analysed individually at the beginning and termination of the test.
pri japonski medaki se trebuh odpre z natančnim prerezom ob ventralni sredinski črti od prsnega obroča do točke, kranialne na anus.
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…).
Jetra se previdno odstranijo z majhno prijemalko in škarjicami;