natgillin/translations-raw huggingface dataset:
The vitamin stock solution is stored frozen in small aliquots.
Za histologijo se cela riba evtanazira in fiksira.
The medium is aerated and stabilised.
Vzorci plazme in ribe se ločeno oštevilčijo, da se ravni VTG povežejo s spolom ribe.
Stock solutions of trace elements for medium M4 and M7
Rezanje ribe za meritev VTG v homogenatu glava/rep in histološko oceno srednje rezine
Stock solutions (I)
Določitev genetskega spola
Amount (mg) made up to 1 litre of deionised water
Pri vrstah, ki imajo ustrezne označevalce, se za določitev genetskega spola odvzame biološki vzorec posamezne ribe.
To prepare the combined stock solution (II):
Pri japonski medaki se odvzame iz predrepne ali hrbtne plavuti.
mix the following amounts (ml) of stock solutions (I) and make up to 1 litre of deionised water
Podroben opis, vključno z vzorčenjem tkiva in določanjem spola z metodo PCR, je naveden v Dodatku 9.
Final concentrations in test solutions (mg/l)
Enako je za zeta opis vzorčenja tkiva in metode PCR za določanje spola naveden v Dodatku 10.
MnCl2·4H2O [1]
Meritev VTG
SrCl2·6H2O [1]
Meritev VTG mora temeljiti na kvantitativno in analitsko validirani metodi.
MoO4·2H2O [1]
Na voljo morajo biti informacije o variabilnosti metode pri preskusu in med preskusi, uporabljene v zadevnem laboratoriju.
CuCl2·2H2O [1]
Vir laboratorijske in medlaboratorijske variabilnosti (najverjetneje) temelji na različnih razvojnih fazah populacije rib.
CaCl2·6H2O
Glede na variabilnost meritev VTG je treba NOEC, ki temeljijo le na tej končni točki, obravnavati zelo previdno.
EDTA·2H2O [1] [2]
Na voljo so različne metode za oceno nastajanja VTG pri vrstah rib, obravnavanih v tem preskusu.
FeSO4·7H2O [1] [2]
Merilna tehnika, ki je razmeroma občutljiva in specifična, je določitev koncentracij proteinov z encimskim imunskim testom (ELISA).
Macro nutrient stock solutions for medium M4 and M7
Uporabiti je treba homologna protitelesa (proti VTG iste vrste) in najpomembnejše homologne raztopine.
Amount made up to 1 litre of deionised water
Določanje spola
Amount of macro nutrient stock solutions added to prepare medium M4 and M7
Glede na postopek vzorčenja VTG se cela riba ali preostali srednji del vsake ribe vstavi v predhodno označeno kaseto za obdelavo in fiksira v ustreznem fiksativu za histološko določitev spola (neobvezno tudi za oceno določanja faze spolnih žlez).
(ml/l)
Smernice za fiksiranje in vstavljanje so navedene v Dodatku 7 in Smernici OECD za določitev histopatologije, povezane z endokrinim sistemom, spolnih žlez pri ribah (22).
Final concentrations in test solutions M4 and M7
Ribe se po obdelavi vstavijo v parafinske bloke.
Vitamin stock solution for medium M4 and M7
Osebki se v parafinski blok vstavijo vzdolžno.
All three vitamin solutions are combined to make a single vitamin stock solution.
Pri vsakem osebku se odvzame vsaj šest vzdolžnih rezin (debeline 3–5 μm) s sprednje ravnine, vključno s spolnim tkivom obeh spolnih žlez.
Amount of vitamin stock solution added to prepare medium M4 and M7
Razmik med temi rezinami mora biti približno 50 μm pri samcih in 250 μm pri samicah.
Thiamine hydrochloride
Ker pa vsak blok pogosto vsebuje samce in samice (če je v vsak blok vstavljen več kot en osebek), mora biti razmik med rezinami iz teh blokov približno 50 μm, dokler se ne pridobi vsaj šest rezin spolnih žlez od vsakega samca.
Cyanocobalamin (B12)
Zato se lahko razmik med rezinami poveča na približno 250 μm pri samicah.
Biotine
Rezine se pobarvajo s hematoksilinom in eozinom ter pregledajo pod svetlobnim mikroskopom, pri čemer se osredotoči na spol (samec, samica, dvospolnik ali nediferencirani osebek).
These substances differ in M4 and M7, as indicated above.
Dvospolnost je opredeljena kot prisotnost več kot enega oocita v testisu na šest analiziranih rezin ali spermatogenih celic (da/ne) v jajčnikih.
These solutions are prepared individually, then poured together and autoclaved immediately.
Histopatologija ter določanje faze jajčnikov in testisov nista obvezna, če pa se izvedeta, je treba rezultate sistematično analizirati in o njih poročati.
Preparation of formulated sediment
Opozoriti je treba, da je za nekatere vrste rib običajna nepopolna razvitost para spolnih žlez, pri čemer je lahko prisotna le ena spolna žleza (npr. japonska medaka in občasno cebrica).
SEDIMENT COMPOSITION
Vsa taka opažanja je treba zabeležiti.
The composition of the formulated sediment should be as follows:
Določitev genetskega spola pri posameznih osebkih japonske medake temelji na prisotnosti ali odsotnosti gena za določitev moškega spola, DMY, ki se nahaja na kromosomu Y.
Sphagnum moss peat, as close to pH 5,5-6,0 as possible, no visible plant remains, finely ground (particle size ≤ 1 mm) and air dried
Genotipski spol medake se lahko določi s sekvenciranjem gena DMY iz DNA, ekstrahirane na primer iz kosa predrepne ali hrbtne plavuti.
4 - 5
Prisotnost DMY kaže posameznika (samca) z geni XY ne glede na fenotip, medtem ko odsotnost DMY kaže posameznika (samico) z geni XX ne glede na fenotip (23).
> 50 % of the particles should be in the range of 50-200 μm
Smernice za pripravo tkiva in metodo PCR so navedene v Dodatku 9.
CaCO3, pulverised, chemically pure
Genetski spol se pri posameznih zetih določa tudi z metodo PCR, ki je opisana v Dodatku 10.
0,05 - 0,1
O pojavu dvospolnosti (za opredelitev glej Dodatek 1) je treba poročati.
Conductivity ≤ 10 μS/cm
Sekundarne spolne značilnosti
PREPARATION
Pri japonski medaki je oblikovanje papil na zadnjem delu predrepne plavuti pri samicah občutljivo na androgen.
The suspension is conditioned for at least two days with gentle stirring at 20 ± 2 °C, to stabilise pH and establish a stable microbial component. pH is measured again and should be 6,0 ± 0,5.
V poglavju C.37 te priloge (38) so zadevne fotografije sekundarnih spolnih značilnosti pri samcih in androgeniziranih samic.
STORAGE
V Dodatku 8 je na voljo diagram poteka za odločitev o uporabi najprimernejšega statističnega preskusa na podlagi značilnosti podatkov, pridobljenih s preskusom.
The dry constituents for preparation of the artificial sediment may be stored in a dry and cool place at room temperature.
Raven statistične značilnosti za vse vključene končne točke je 0,05.
The formulated (wet) sediment should not be stored prior to its use in the test.
Deleži spola in genetskega spola
It should be used immediately after the 7 days conditioning period that ends its preparation.
Če obstaja monotoni odziv na odmerek, je treba deleže spola za znatne učinke (pristop NOEC/LOEC) izpostavljenosti analizirati s preskusom po Jonckheere-Terpstraju (preskus trenda).
OECD (1984), Earthworm, Acute Toxicity Test, Test Guideline No. 207, Guidelines for the Testing of Chemicals, OECD, Paris.
Če je ugotovljena nemonotonost, je treba uporabiti preskus med pari:
Meller, M., Egeler, P., Roembke, J., Schallnass, H., Nagel, R. and B. Streit (1998), Short-term toxicity of lindane, hexachlorobenzene and copper sulfate on tubificid sludgeworms (Oligochaeta) in artificial media, Ecotox.
Dunnettov preskus se uporabi, če se lahko pridobita normalnost in homogenost varianc.
Environ.
Tamhane-Dunnetov preskus se uporabi, če je prisotna homogenost varianc.
Safety, 39:
Sicer se uporabi Mann-Whitneyjev preskus z Bonferroni-Holmovo prilagoditvijo.
10-20.
V Dodatku 8 je diagram poteka, v kateri so navedeni statistični podatki o deležih spola.
Chemical Characteristics of an Acceptable Dilution water
Deleže spola je treba v tabelah predstaviti kot koncentracijski delež ± standardni odklon samcev, samic, dvospolnikov in nediferenciranih osebkov.
CONSTITUENT
Statistično značilnost je treba poudariti.
CONCENTRATIONS
Primeri so navedeni v validacijskem poročilu faze 2 preskusa spolnega razvoja rib (42).
Hardness as CaCO3
Genetski spol je treba sporočiti kot delež sprememb fenotipičnega spola samic, samcev, dvospolnikov in nediferenciranih osebkov.
However, it should be noted that if there is an interaction suspected between hardness ions and the test chemical, lower hardness water should be used (and thus, Elendt Medium M4 should not be used in this situation).
Koncentracije VTG
Guidance for test performance
Analizirati je treba pomemben učinek (pristop NOEC/LOEC) izpostavljenosti koncentracijam VTG.
Example of a breeding cage:
Bolj zaželen kot t-preskus z Bonferronijevim popravkom je Dunnettov preskus.
A gauze on the top and at least one side of the cage (mesh size ca.
Če se uporabi Bonferronijev popravek, je bolj zaželen Bonferroni-Holmov popravek.
1 mm)
Dovoliti je treba logaritmično transformacijo VTG, da se dosežeta normalnost in homogenost varianc.
B aperture for placing the emerged adults inside the breeding cage and to remove the laid egg ropes from the crystallisation dishes (not shown in this graphic)
Če je odziv na koncentracijo skladen z monotonostjo, ima preskus po Jonckheere-Terpstraju prednost pred vsemi navedenimi preskusi.
C breeding cage size minimum 30 cm length, 30 cm height and 30 cm width
Če se uporabi t-preskus ali Dunnettov preskus, za nadaljevanje ni potreben F-preskus pomembnosti analize variance.
Example of a test vessel:
Podrobnosti so navedene v tabeli poteka v Dodatku 8.
A pasteur pipette for air supply of the overlying water
Rezultate je treba predstaviti v tabelah kot koncentracijske sredine ± standardni odklon samcev, samic, dvospolnikov in nediferenciranih osebkov.
B glass lid to prevent emerged midges from escaping
Poudariti je treba statistično značilnost za fenotipične samice in fenotipične samce.
C water surface layer
Dejanske koncentracije preskusne kemikalije
D test vessel (glass beaker minimum 600 ml)
Dejanske koncentracije preskusne kemikalije v komorah je treba analizirati tako pogosto, kot je opisano v odstavku 34.
E sediment layer
Rezultate je treba sporočiti v tabelah kot srednjo koncentracijo ± standardni odklon na podlagi ponovitev in na podlagi koncentracij skupaj s poudarjenimi informacijami o številu vzorcev in izjemami pri srednji koncentraciji za tretiranje ± 20 %.
Example of an exhauster for capturing adult midges (arrows indicate air flow direction):
Primeri so navedeni v validacijskem poročilu faze 2 preskusa spolnega razvoja rib (42).
A glass tube (inner diameter ca.
Rezultate preskusa je treba razlagati previdno, kadar so merjene koncentracije preskusne kemikalije v preskusnih raztopinah blizu meje zaznavnosti analitske metode.
5 mm) connected to a self-priming pump B cork of vulcanised rubber, perforated with glass tube (A).
nominalne preskusne koncentracije, srednje vrednosti izmerjenih vrednosti in njihovi standardni odkloni v preskusnih komorah in metoda, s katero so bile te dobljene (uporabljeno analitsko metodo je treba predstaviti v prilogi), ter dokazi, da se meritve nanašajo na koncentracije preskusne kemikalije v pravi raztopini,
On the inside, the opening of glass tube (A) is covered with some cotton and a gauze (mesh size ca.
podrobne informacije o hranjenju (npr. vrsta hrane, vir, dana količina in pogostost dajanja ter analiza onesnaževal (npr. PCB, PAH in organoklorni pesticidi), če je ustrezno.
1 mm) to prevent damaging the midges when they are sucked into the exhauster
Dokazi, da so kontrole izpolnjevale merila za veljavnost:
C transparent container (plastic or glass, length ca.
podatke o hitrosti izvalitve je treba predstaviti v tabelah kot delež na ponovitev in na koncentracijo.
15 cm) for captured midges
Poudariti je treba izjeme pri merilih sprejemljivosti (v kontrolah).
D cork of vulcanised rubber, perforated with tube (E).
Preživetje je treba predstaviti kot delež na ponovitev in na koncentracijo.
To release midges into the breeding cage, cork D is released from container C
Poudariti je treba izjeme pri merilih za veljavnost (v kontrolah),
E tube (plastic or glass, inner diameter ca.
jasna navedba rezultatov, pridobljenih na različnih opazovanih končnih točkah:
8 mm) to collect adult midges from vessel
preživetje embriov in uspešna izvalitev;
Schematic presentation of a life-cycle test:
meritve VTG (ng/g homogenata, ng/ml plazme ali ng/mg jeter);
A 1st generation — test vessels containing a sediment-water system, eight replicates, 20 first instar larvae per vessel
Rezultate preskusa je treba predstaviti kot srednje vrednosti ± standardni odklon (SD) ali standardno napako (SE).
B four test vessels for each breeding cage, A and B
Statistične podatke je treba sporočiti vsaj kot NOEC in LOEC ter intervale zaupanja.
C breeding cages (A and B) for swarming, mating and oviposition
Upoštevati je treba statistično tabelo poteka (Dodatek 8).
D crystallising dishes for deposition of egg ropes
‚Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals‘, Environmental Toxicology and Chemistry 15, str. 194–202.
E micro plates, one well for each egg rope
Sumpter, J. P., in Jobling, S. (1995). ‚Vitellogenesis As A Biomarker for Estrogenic Contamination of the Aquatic Environment‘, Environmental Health Perspectives 103, str. 173– 178.
F 2nd generation — test vessels containing a sediment-water system, eight replicates, 20 first instar larvae per vessel
‚An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin‘, Environmental Toxicology and Chemistry 18, str. 337–347.
C.41.
‚Development of an ELISA for vitellogenin in whole body homogenate of zebrafish (Danio rerio)‘, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 130, str. 119– 131.
FISH SEXUAL DEVELOPMENT TEST
Andersen, L., Bjerregaard, P., in B. Korsgaard (2003). ‚Vitellogenin induction and brain aromatase activity in adult male and female zebrafish exposed to endocrine disrupters‘, Fish Physiology and Biochemistry 28, str. 319–321.
This test method is equivalent to OECD test guideline (TG) 234 (2011).
‚Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone‘, Aquatic Toxicology 65, str. 397–411.
It is based on a decision from 1998 to develop new or update existing test methods for the screening and testing of potential endocrine disrupters.
‚Utility of a juvenile fathead minnow screening assay for detecting (anti-)estrogenic substances‘, Environmental Toxicology and Chemistry 21, str. 319–326.
The Fish Sexual Development Test (FSDT) was identified as a promising test method covering a sensitive fish life stage responsive to both oestrogen and androgen-like chemicals.
‚Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults‘, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 145, str. 533–541.
The test method went through an inter-laboratory validation exercise from 2006 to 2010, where Japanese medaka (Oryzias latipes), zebrafish (Danio rerio) and three spined stickleback (Gasterosteus aculeatus) were validated and fathead minnow (Pimephales promelas) was partially validated (41) (42) (43).
‚Fathead minnow (Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds‘, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 123, str. 113– 125.
This protocol includes Japanese medaka, the three-spined stickleback and zebrafish.
‚Development and validation of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio rerio)‘, Environmental Toxicology and Chemistry 21, str. 1699– 1708.
The protocol is in principle an enhancement of OECD TG 210 Fish, Early Life Stage Toxicity Test (1), where the exposure is continued until the fish are sexually differentiated, i.e. about 60 days post-hatch (dph) for Japanese medaka, the three-spined stickleback and zebrafish (the exposure period can be shorter or longer for other species that are validated in the future), and endocrine-sensitive endpoints are added.
‚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, str. 161– 169.
The combination of the two core endocrine endpoints, vitellogenin (VTG) concentration and phenotypic sex ratio enable the test to indicate the mode of action of the test chemical.
Tatarazako, N., Koshio, M., Hori, H., Morita, M.. in Iguchi, T. (2004). ‚Validation of an enzyme-linked immunosorbent assay method for vitellogenin in the medaka‘, Journal of Health Science 50, str. 301–308.
Due to the population-relevant change in phenotypic sex ratio, the FSDT can be used for hazard and risk assessment.
Eidem, J. K., Kleivdal, H., Kroll, K., Denslow, N., van Aerle, R., Tyler, C., Panter, G., Hutchinson, T., in Goksoyr, A. (2006). ‚Development and validation of a direct homologous quantitative sandwich ELISA for fathead minnow (Pimephales promelas) vitellogenin.
However, if the test is used for hazard or risk assessment, the stickleback should not be used because the validation data available so far showed that in this species the alterations of phenotypic sex ratio by the test chemicals were uncommon.
Aquatic Toxicology‘, 78, str. 202–206.
The protocol is based on fish exposed via water to chemicals during the sex labile period in which the fish is expected to be most sensitive to the effects of endocrine disrupting chemicals that interfere with sexual development.
‚Evaluation of a commercial kit for measuring vitellogenin in the fathead minnow (Pimephales promelas)‘, Ecotoxicology and Environmental Safety 64, str. 101– 105.
Two core endpoints are measured as indicators of endocrine-associated developmental aberrations, the VTG concentrations and sex ratios (proportions of sex) determined via gonad histology.
Holbech, H., Petersen, G. I., Norman, A., Örn, S., Norrgren, L., in Bjerregaard, P. (2001b). ‚Suitability of zebrafish as test organism for detection of endocrine disrupting chemicals.
Gonadal histopathology (evaluation and staging of oocytes and spermatogenetic cells) is optional.
Comparison of vitellogenin in plasma and whole body homogenate from zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss)‘, Nordic Council of Ministers, TemaNord 2001:597, str. 48–51.
Additionally, the genetic sex is determined whenever possible (e.g. in Japanese medaka and the three spined stickleback).
‚Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening‘, Analytical and Bioanalytical Chemistry 378, str. 621–633.
The presence of a genetic sex marker is a considerable advantage as it increases the power of the sex ratio statistics and enables the detection of individual phenotypic sex reversal.
Orn, S., Yamani, S. in Norrgren, L. (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, str. 237–243.
Other apical endpoints that should be measured include hatching rate, survival, length and body weight.
Scholz, S., in Kluver, N. (2009). ‚Effects of Endocrine Disrupters on Sexual, Gonadal Development in Fish, Sexual Development‘ 3, str. 136– 151.
The test method might be adaptable to other species than those mentioned above provided that the other species undergo a validation equal to the one accomplished for Japanese medaka, the three-spined stickleback and zebrafish, that the control fish are sexually differentiated at the end of the test, that VTG levels are sufficiently high to detect significant chemical-related variations, and that sensitivity of the test system is established using endocrine active reference chemicals ((anti)-oestrogens, (anti)-androgens, aromatase inhibitors etc).
Fenske, M., Maack, G., Schafers, C. in Segner, H. (2005). ‚An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio‘, Environmental Toxicology and Chemistry 24, str. 1088– 1098.
In addition, any validation report(s) referring to FSDT data using other species should be reviewed by the OECD, and the validation outcome should be considered as satisfactory.
‚Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes‘, Developmental Dynamics 231, str. 518–526.
Initial considerations and limitations
Shinomiya, A., Otake, H., Togashi, K., Hamaguchi, S., in Sakaizumi M. (2004), ‚Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations‘, Zoological Science 21, str. 613–619.
VTG is normally produced by the liver of female oviparous vertebrates in response to circulating endogenous oestrogen (2).
‚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, str. 8897–8901.
The VTG synthesis is very limited, though detectable, in immature fish and adult male fish because they lack sufficient circulating oestrogen.
P., Evans, R. E., Wautier, K. G., Mills, K. H., Blanchfield, P. J., Park, B. J., Baron, C. L., in Kidd, K. A. (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, str. 1920– 1935.
However, the liver is capable of synthesising and secreting VTG in response to exogenous oestrogen stimulation (3) (4) (5).
‚Development of chronic tests for endocrine active chemicals – Part 1.
The measurement of VTG serves for the detection of chemicals with oestrogenic, anti-oestrogenic, androgenic modes of action and chemicals that interfere with steroidogenesis as for example aromatase inhibitors.
An extended fish early-life stage test for oestrogenic active chemicals in the fathead minnow (Pimephales promelas)‘, Aquatic Toxicology 77, str. 279–290.
The detection of oestrogenic chemicals is possible via the measurement of VTG induction in male fish, and it has been abundantly documented in the scientific peer-reviewed literature.
‚Detection of endocrine disrupters: Evaluation of a Fish Sexual Development Test (FSDT)‘, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 144, str. 57–66.
VTG induction has also been demonstrated following exposure to aromatisable androgens (6) (7).
Andersen, L., Kinnberg, K., Holbech, H., Korsgaard, B., in Bjerregaard, P. (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, str. 257–266.
A reduction in the circulating level of oestrogen in females, for instance through the inhibition of the aromatase converting the endogenous androgen to the natural oestrogen 17β-oestradiol, causes a decrease in the VTG concentration, which is used to detect chemicals having aromatase inhibiting properties or steroidogenesis inhibitors more broadly (33).
Morthorst, J. E., Holbech, H., in Bjerregaard, P. (2010). ‚Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations‘, Aquatic Toxicology 98, str. 336–343.
The biological relevance of the VTG response following oestrogenic/aromatase inhibition is established and has been broadly documented (8) (9).
‚Effects of the antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes)‘, Aquatic Toxicology 63, str. 391–403.
However, it is possible that production of VTG in females can also be affected by general toxicity and non-endocrine toxic modes of action.
‚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, str. 11–21.
Several measurement methods have been successfully developed and standardised for routine use to quantify VTG in blood, liver, whole body or head/tail homogenate samples collected from individual fish.
‚Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio)‘, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 145, str. 165– 170.