natgillin/translations-raw huggingface dataset:
The FSDT validation phases (1 and 2) did cover oestrogenic, androgenic and steroidogenesis inhibiting chemicals.
Nagahama (2004), ’Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes’, Developmental Dynamics 231, pp.
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.
518–526.
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.
Shinomiya, A., H. Otake, K. Togashi, S. Hamaguchi, and M. Sakaizumi (2004), ’Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations’, Zoological Science 21, pp.
The test conditions should be flow-through unless not possible due to the availability or nature (e.g. limited solubility) of the test chemical.
613–619.
The test starts with the placing of newly fertilised eggs (before cleavage of the blastodisc) in the test chambers.
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, pp.
The loading of the chambers is described for each species in paragraph 27.
8897–8901.
For the validated fish species, Japanese medaka, the three-spined stickleback and zebrafish, the test is terminated at 60 dph.
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, pp.
At test termination, all fish are euthanised humanely.
1920–1935.
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;
Tyler (2006), ’Development of chronic tests for endocrine active chemicals – Part 1.
optionally, histopathology (e.g. staging of gonads, severity of intersex) can be performed.
An extended fish early-life stage test for oestrogenic active chemicals in the fathead minnow (Pimephales promelas)’, Aquatic Toxicology 77, pp.
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).
279–290.
An overview of relevant test conditions specific for validated species:
Petersen, P. Jackson, K. Hylland, L. Norrgren, and P. Bjerregaard (2006), ’Detection of endocrine disrupters: Evaluation of a Fish Sexual Development Test (FSDT)’, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 144, pp.
Japanese medaka, the three-spined stickleback and zebrafish is provided in Appendix 2.
57–66.
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.
Andersen, L., K. Kinnberg, H. Holbech, B. Korsgaard, and 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, pp.
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.
257–266.
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).
Morthorst, J.E., H. Holbech, and P. Bjerregaard (2010), ’Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations’, Aquatic Toxicology 98, pp.
Test acceptance criteria
336–343.
The dissolved oxygen concentration should be at least 60 per cent of the air saturation value (ASV) throughout the test;
Metcalf (2003), ’Effects of the antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes)’, Aquatic Toxicology 63, pp.
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);
391–403.
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;
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, pp.
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;
11–21.
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):
Petersen, and P. Bjerregaard (2007), ’Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio)’, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 145, pp.
Japanese medaka
165–170.
Three-spined stickleback
Tämän liitteen luku C.14 Nuorten kalojen kasvutesti.
Growth
Tämän liitteen luku C.4 Nopea biohajoavuus.
Fish wet weight, blotted dry
Tämän liitteen luku C.37 21 päivän mittainen seulontatutkimus kaloilla:
> 75 mg
Lyhytaikainen estrogeeni- ja androgeeniaktiivisuuteen sekä aromataasin estoon kohdistuva seulonta.
Length (standard length)
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 pp 768–779.
> 20 mm
Lyhenteet ja määritelmät
> 14 mm
Apikaalinen päätepiste
Sex ratio (% males or females)
aiheuttaa vaikutuksen populaation tasolla
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.
air saturation value, ilman kyllästysarvo
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.
Biomarkkeri
Test chambers
aiheuttaa vaikutuksen yksilön tasolla
Any glass, stainless steel or other chemically inert chambers can be used.
aine tai seos
The dimensions of the chambers should be large enough to allow compliance with loading rate criteria given below.
days post hatch, (xx) päivää kuoriutumisen jälkeen
It is desirable that test chambers be randomly positioned in the test area.
Y-kromosomispesifi DM-domeenin geeni, jota medakakalojen kehittyminen koiraiksi edellyttää.
A randomised block design with each concentration being present in each block is preferable to a completely randomised design.
entsyymivälitteinen immunosorbenttimääritys
The test chambers should be shielded from unwanted disturbance.
Kalan paino
Recommended fish species are given in Appendix 2.
kalan märkäpaino (kuivattu imeyttämällä vesi paperiin)
The procedures for inclusion of new species are given in paragraph 2.
Fish Sexual Development Test, kalojen sukupuolikehitystä tutkiva testi
Holding of parental fish
hypotalamus-aivolisäke-sukurauhasakseli
Details on holding the parental fish under satisfactory conditions may be found in OECD TG 210(1).
Sekasukupuolinen kala
Parental fish should be fed once or twice a day with appropriate food.
kala, jolla on kuutta analysoitua leikettä kohti useampi kuin yksi oosyytti kiveksissä tai spermatogeneettisiä soluja munasarjoissa (kyllä/ei)
Handling of embryos and larvae
kalojen märkäpaino tilavuusyksikköä kohti
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.
mode of action, vaikutustapa
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.
Reverse Transcriptase Polymerase Chain-Reaction, käänteiskopioijaentsyymi-polymeraasiketjureaktio
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.
Eriytymätön kala
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.
kala, jonka sukupuolirauhasissa ei ole selvästi näkyviä itusoluja
The timing of this transfer varies with the species and transfer may not always be necessary.
vitellogeniini
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.
FSDT-testin (makean veden lajit) koeolosuhteet
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.
Medaka (Oryzias latipes)
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.
Seeprakala (Danio rerio)
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.
Kolmipiikki (Gasterostreus aculeatus)
Details about chemical analysis and water collection can be found in paragraph 34.
Läpivirtaus tai puolistaattinen
Flow-through system should be used if practically possible.
Loistelamppu (laajakirjoinen)
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.
Valovoima
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.
10–20 μE/m2/s, 540–1080 luksia tai 50–100 ft-c:tä (ympäröivän laboratorion tasot)
A flow rate equivalent to at least five test chamber volumes per 24 hours has been found suitable (1).
12–16 h valoisaa, 8–12 h pimeää
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.
Astian vähimmäiskoko
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).
Yksittäisiin astioihin on mahduttava vähintään seitsemän litraa vettä
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).
Testiorganismien ikä altistuksen alussa
The use of solvents should be avoided but may be necessary in some cases in order to produce a suitably concentrated stock solution.
Hiljattain hedelmöityneet munat (varhainen rakkula-aste)
Examples of suitable solvents are given in (36).
Hiljattain hedelmöityneet munat
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).
Munien lukumäärä käsittelyä kohti
For the semi-static technique, two different renewal procedures may be followed.
Vähintään 120
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.
Vähintään 3 (sekä asianmukaiset kontrollit)
Conditions of Exposure
Rinnakkaisnäytteiden lkm käsittelyä kohti
Collection of eggs and duration
Vähintään 4 (ellei käytetty neliöjuureen perustuvaa jakamista kontrolliryhmiin)
To avoid genetic bias, eggs are collected from a minimum of three breeding pairs or groups, mixed and randomly selected to initiate the test.
Elävä ravinto (Artemia), pakastetut täysikasvuiset suolalehtijalkaiset, hiutaleruoka jne. Kalat suositellaan ruokittavan kahdesti päivässä.
For the three-spined stickleback, see the description of artificial fertilisation in Appendix 11.
Kalanpoikasten erikoisruoka, elävä ravinto (Artemia), pakastetut täysikasvuiset suolalehtijalkaiset, hiutaleruoka jne. Kalat suositellaan ruokittavan kahdesti päivässä.
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.
Ei tarpeen, ellei liuenneen hapen pitoisuus ole alle 60 % ilman kyllästysarvosta
The test should continue until sexual differentiation in the control group is completed (60 dph for Japanese medaka, the three-spined stickleback and zebrafish).
Ei tarpeen, ellei liuenneen hapen pitoisuus ole alle 70 % ilman kyllästysarvosta
Loading
Puhdas pinta- tai kaivovesi tai synteettinen vesi
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).
Testikemikaalialtistuksen kesto 60 pv kuoriutumisen jälkeen
The eggs should be randomly distributed (by using statistical tables for randomisation) among treatments.
Kuoriutumisen onnistuminen, eloonjäämisen morfologia, VTG sukup.rauhasten histologia, geneettinen sukupuoli sukup.jakauma
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.
Kuoriutumisen onnistuminen, eloonjäämisen morfologia, VTG sukup.rauhasten histologia, sukup.jakauma
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.
Testin hyväksymiskriteerit (kontrollien yhdistetyt rinnakkaisnäytteet)
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.
Kuoriutumisen onnistuminen > 80 %
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.
Kuoriutumisen jälkeinen eloonjääminen ≥ 70 %,
The photoperiod and water temperature should be appropriate for the test species (see Appendix 2 for experimental conditions for the FSDT).
kasvu (kalan märkäpaino, kuivattu imeyttämällä vesi paperiin) > 150 mg
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.
Pituus (vakiopituus) > 20 mm
Surplus food and faeces should be removed, as necessary, to avoid accumulation of waste.
Pituus (vakiopituus) > 14 mm
As experience is gained, food and feeding regimes are continuously being refined to improve survival and optimise growth.
Pituus (vakiopituus) > 20 mm
Effort should therefore be made to confirm the proposed regime with acknowledged experts.
Sukup.jakauma (% koiraita tai naaraita) 30–70 %
Feeding should be withheld 24 hours before ending the test.
Hyväksyttävän laimennusveden kemialliset ominaisuudet
Examples of appropriate food items are listed in Appendix 2 (see also the OECD Fish Testing Framework (39).
KESKITTYMÄ
Test chemicals should be spaced as described in Appendix 4.
Hiukkaset
A minimum of three test concentrations in at least four replicates should be used.
Testimenetelmästä C.14 / Ohjeita testipitoisuuksista
The curve relating LC50 to period of exposure in the acute studies available should be considered when selecting the range of test concentrations.
Sarake (välillä 100–10 tai välillä 10–1 olevien pitoisuuksien lukumäärä) [1]
Five test concentrations are recommended if the data are to be used for risk assessment.
Sarakkeesta voi valita kolmen (tai useamman) peräkkäisen pitoisuuden sarjan.
Concentrations of the chemical higher than 10 % of the acute adult LC50 or 10 mg/l, whichever is the lower, need not be tested.
Sarakkeessa (x) olevien pitoisuuksien keskivälillä olevia arvoja on sarakkeessa (2x + 1).
The maximum test concentration should be 10 % of the LC50 on the larval/juvenile life-stage.
Luetellut arvot voivat olla prosenttiosuuksina tilavuusyksikköä tai painoyksikköä kohti ilmaistuja pitoisuuksia (mg/l tai μg/l).
Controls
Arvot voidaan tarvittaessa jakaa tai kertoa kymmenen potensseilla.
A dilution water control (≥ 4 replicates) and, if relevant, a solvent control (≥ 4 replicates) should be run in addition to the test concentrations.
Saraketta 1 voidaan käyttää, jos myrkyllisyystaso on hyvin epävarma.
Only solvents that have been investigated not to have any statistical significant influence on the test endpoints should be used in the test.
Ohjeet pään ja pyrstön homogenisointiin (nuoret seeprakalat, rasvapäämutut, kolmipiikit ja medakat)
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.
Tässä kohdassa kuvataan menettelyt, jotka edeltävät vitellogeniinipitoisuuden kvantifiointia.
However, every effort should be made to avoid the use of such solvent or keep solvent's concentrations to a minimum.
Myös muita menettelyjä, joiden tuloksena on samankaltainen vitellogeniinin kvantifiointi, voidaan käyttää.
Frequency of Analytical Determinations and Measurements
Testissä voidaan valita, määritetäänkö vitellogeniinipitoisuus veren plasmasta tai maksasta pää-/pyrstöhomogenaatin sijasta.
Chemical analysis of the test chemical concentration should be performed before initiation of the test to check compliance with the acceptance criteria.
Kalan pää ja pyrstö leikataan irti testikuvauksessa esitetyllä tavalla.
All replicates should be analysed individually at the beginning and termination of the test.
Kaikki dissekointivälineet ja leikkauspöytä on huuhdeltava ja puhdistettava kunnolla (esimerkiksi 96-prosenttisella etanolilla) jokaisen kalan käsittelyn jälkeen, jottei naaraiden tai niiden koiraiden, joille kemikaali on aiheuttanut vaikutuksia, vitellogeniini saastuta terveitä koiraita.
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…).
Kun homogeeninen massa on valmis, siihen lisätään 4–10 kertaa kudoksen painon verran jääkylmää homogenisointipuskuria (muista laimennus).
If samples are stored to be analysed at a later time, the storage method of the samples should be previously validated.
Näytteet laitetaan jäähän siihen saakka, kunnes ne sentrifugoidaan 4 °C:ssa 50000 g:ssa 30 minuutin ajan.
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.
Laita pipetillä 20–50 μl:n supernatanttiannoksia (huomaa määrä) vähintään kahteen putkeen upottamalla pipetin kärki pinnan rasvakerroksen läpi ja vetämällä varovasti supernatanttia pipettiin ilman rasva- tai pellettifraktioita.
During the test, dissolved oxygen, pH, total hardness, conductivity, salinity (if relevant), and temperature should be measured in all test chambers.
Pidä puskuri jään päällä käytön aikana.
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.
12 ml Tris-HCl pH 7,4 + 120 μl proteaasinestäjäsekoitus (tai vastaavat proteaasinestäjäsekoitukset).TRIS:
Temperature should preferably be monitored continuously in at least one test chamber.
TRIS-ULTRA PURE (ICN)Proteaasinestäjäsekoitus:
Results should be based on measured concentrations.
Sigman (nisäkkäiden kudoksille tarkoitettu) tuote nro P 8340.
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.
Ohjeet seeprakalojen (Danio rerio) pää- ja pyrstöhomogenaatin vitellogeniinipitoisuuden kvantifiointiin (muokattu Holbechin ja muiden (2001) laatimista ohjeista) myös muita menetelmiä, joissa käytetään homologisia vasta-aineita ja standardeja, voidaan käyttää.
Observations and measurements
Mikrotitterilevyt (sertifioitu Maxisorp F96, Nunc, Roskilde, Tanska), jotka on aiemmin päällystetty 5 μg/ml:lla anti-seeprakala-lipovitelliini-IgG:tä, sulatetaan ja pestään kolme kertaa pesupuskuriliuoksella (*).
Stage of embryonic development
Puhdistettu seeprakalan vitellogeniinistandardi [1] laimennetaan asteittain pitoisuuteen 0,2, 0,5, 1, 2, 5, 10 ja 20 ng/ml laimennuspuskurissa (**), ja näytteet laimennetaan vähintään 200 kertaa (matriisivaikutuksen estämiseksi) laimennuspuskurissa, jonka jälkeen ne laitetaan mikrotitterilevyihin.
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.
Testikontrollia laitetaan kaksinkertainen määrä.
Hatching and survival
Jokaiseen kuoppaan laitetaan 150 μl.
Observations on hatching and survival should be made at least once daily and numbers recorded.
Standardit laitetaan kaksinkertaisesti ja näytteet kolminkertaisesti.
Dead embryos, larvae and juvenile fish should be removed as soon as observed since they can decompose rapidly and may be broken up by the actions of the other fish.
Anna inkuboitua ravistimessa yön yli 4 °C:n lämpötilassa.
Extreme care should be taken when removing dead individuals not to knock or physically damage adjacent eggs/larvae, these being extremely delicate and sensitive.
Levyt pestään viisi kertaa pesupuskurilla.
Criteria for death vary according to life stage:
Dekstraaniketjuun liittynyt HRP (esimerkiksi AMDEX A/S, Tanska) ja konjugoituneet vasta-aineet laimennetaan pesupuskurissa;
for eggs:
varsinainen laimennus vaihtelee erän ja iän mukaan.
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;
Jokaiseen kuoppaan laitetaan 150 μl, ja levyjä inkuboidaan ravistimessa tunnin ajan huoneenlämmössä.
for larvae and juvenile fish:
Levyt pestään viisi kertaa pesupuskurilla, ja niiden pohja puhdistetaan huolellisesti etanolilla.
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.
Jokaiseen kuoppaan laitetaan 150 μl TMB plus -substraattia (***).
Abnormal appearance
Suojaa levy valolta foliolla ja tarkkaile värin kehittymistä ravistimessa.