natgillin/translations-raw huggingface dataset:
Adult midges in the breeding cage need no feeding normally, but fecundity and fertility are enhanced when a cotton wool pad soaked in a saturated sucrose solution is offered as a food source for emerged adults (34).
Med letoma 2006 in 2010 se je izvajala medlaboratorijska validacija preskusne metode, pri čemer so bile validirane japonska medaka (Oryzias latipes), cebrica (Danio rerio) in zet (Gasterosteus aculeatus), črnoglavi pisanec (Pimephales promelas) pa je bil delno validiran (41) (42) (43).
Gentle aeration of the overlying water in the test vessels is supplied 24 hours after addition of the first instar larvae of both generations and is continued throughout the test (care should be taken that the dissolved oxygen concentration does not fall below 60 % of ASV).
Ta protokol vključuje japonsko medako, zeta in cebrico.
Aeration is provided through a glass Pasteur pipette of which the outlet is fixed 2-3 cm above the sediment layer giving a few bubbles/sec.
Protokol je načeloma razširitev OECD TG 210 Ribe, preskus strupenosti v zgodnji življenjski fazi (1), kjer se izpostavljenost nadaljuje, dokler niso ribe spolno diferencirane, tj. približno 60 dni po tem, ko se izvalijo (DPH), pri japonski medaki, zetu in cebrici (obdobje izpostavljenosti je lahko pri drugih vrstah, ki še bodo validirane, krajše ali daljše), pri čemer se dodajo endokrine občutljive končne točke.
When testing volatile chemicals, consideration should be given not to aerate the sediment-water system, while at the same time the validity criterion of minimal 60 % ASV (paragraph 10) should be fulfilled.
S preskusom spolnega razvoja rib se ocenjujejo učinki v zgodnji življenjski fazi in možne škodljive posledice kemikalij, ki domnevno povzročajo endokrine motnje (npr. inhibitorji estrogenov, androgenov in steroidogeneze) spolnega razvoja.
Further guidance is provided in (16).
Kombinacija dveh glavnih endokrinih končnih točk, koncentracija vitelogenina (VTG) in fenotipsko razmerje med spoloma omogočajo, da se s preskusom nakaže način delovanja preskusne kemikalije.
The test with C. riparius is conducted at a constant temperature of 20 °C (± 2 °C).
Zaradi spremembe fenotipskega razmerja med spoloma, ki je pomembna za populacijo, se lahko preskus spolnega razvoja rib uporablja za oceno nevarnosti in tveganja.
For C. dilutus and C. yoshimatsui, recommended temperatures are 23 °C and 25 °C (± 2 °C), respectively.
Če se preskus uporabi za oceno nevarnosti ali tveganja, pa se zet ne sme uporabiti, ker validacijski podatki, ki so do zdaj na voljo, kažejo, da so pri tej vrsti spremembe fenotipskega razmerja med spoloma, ki jih povzroči preskusna kemikalija, neobičajne.
A 16 hours photoperiod is used and the light intensity should be 500 to 1000 lux.
Protokol temelji na ribah, ki so kemikalijam izpostavljene prek vode v spolno nestabilnem obdobju, v katerem naj bi bile ribe najbolj občutljive na učinke kemikalij, ki povzročajo endokrine motnje, ki ovirajo spolni razvoj.
For the breeding cages an additional one hour dawn and dusk phase may be included.
Glavni končni točki se izmerita kot kazalnika razvojnih kromosomskih aberacij, povezanih z endokrinim sistemom, koncentracije VTG in razmerja med spoloma (deleža po spolu), določena s histologijo spolnih žlez.
Exposure duration
Histopatologija spolnih žlez (ocena in določanje faze oocitov in spermatogenih celic) ni obvezna.
Spiked water design:
Poleg tega se določi genetski spol, kadar koli je mogoče (npr. pri japonski medaki in zetu).
The exposure period of the 1st generation starts when the test chemical is spiked into the overlying water of the test vessels (which is one day after insertion of the larvae — for possible amendments of the exposure design, see paragraph 7).
Prisotnost označevalca genetskega spola je pomembna prednost, ker poveča moč statističnih podatkov o razmerju med spoloma in omogoči zaznavanje posamezne spremembe fenotipičnega spola.
Exposure of the 2nd larval generation starts immediately, since they are inserted into a sediment-water system that has been already spiked.
Druge zgornje končne točke, ki jih je treba izmeriti, vključujejo hitrost izvalitve, preživetje, dolžino in telesno težo.
The maximum exposure duration for the 1st generation is 27 days and for the 2nd generation 28 days (the 1st generation larvae spend one day in the vessels without exposure) for C. riparius and C. yoshimatsui.
Preskusna metoda bi se lahko prilagodila drugim vrstam, ki niso omenjene zgoraj, če bi prestale enako validacijo, kot so jo japonska medaka, zet in cebrica, če so ribe iz kontrole ob koncu preskusa spolno diferencirane, če so ravni VTG dovolj visoke za zaznavanje pomembnih variacij, povezanih s kemikalijo, in če se z referenčnimi kemikalijami z endokrinim delovanjem ((anti-)estrogeni, (anti-)androgeni, inhibitorji aromataze itd.) vzpostavi občutljivost preskusnega sistema.
Considering the overlap, the complete test duration is approximately 44 days.
Poleg tega mora OECD pregledati vsa validacijska poročila, ki se nanašajo na podatke o preskusu spolnega razvoja rib z uporabo drugih vrst, rezultat validacije pa se mora šteti za zadovoljivega.
For C. dilutus, maximum exposure durations are 64 and 65 days, for the 1st and 2nd generation, respectively.
Začetni pomisleki in omejitve
The total duration is approximately 100 days.
VTG običajno proizvajajo jetra samic oviparnih vretenčarjev kot odziv na kroženje endogenega estrogena (2).
Spiked sediment design:
Sinteza VTG je pri nezrelih ribah in odraslih samcih rib zelo omejena, ker nimajo dovolj krožečega estrogena, čeprav zaznavna.
exposure starts with the addition of the larvae and is maximum 28 days for both generations for C. riparius and C. yoshimatsui and maximum 65 days for both generations for C. dilutus.
Vendar so jetra sposobna sinteze in izločanja VTG kot odziv na spodbujanje eksogenega estrogena (3) (4) (5).
Emergence
Meritev VTG je namenjena zaznavanju kemikalij z estrogenimi, antiestrogenimi in androgenimi načini delovanja ter kemikalijam, ki vplivajo na steroidogenezo, kot so na primer inhibitorji aromataze.
Development time and the total number of fully emerged and alive male and female midges are determined for both generations.
Zaznavanje estrogenih kemikalij je mogoča z meritvijo indukcije VTG pri samcih in je bila obsežno dokumentirana v strokovno pregledani znanstveni literaturi.
Males are easily identified by their plumose antennae and thin body posture.
Indukcija VTG je bila dokazana tudi po izpostavljenosti androgenom, ki se lahko aromatizirajo (6) (7).
Test vessels of both generations should be observed at least three times per week to make visual assessment of any abnormal behaviour of the larvae (e.g. leaving sediment, unusual swimming), compared to the control.
Zaradi zmanjšanja ravni kroženja estrogena pri samicah, na primer z zaviranjem aromataze, ki spremeni endogeni androgen v naravni estrogen 17β-estradiol, se zmanjša koncentracija VTG, ki se uporablja za zaznavanje kemikalij, ki imajo lastnosti zaviranja aromataze, ali širše inhibitorjev steroidogeneze (33).
During the period of emergence, which starts about 12 days after insertion of the larvae for C. riparius and C. yoshimatsui (after 20 days for C. dilutus), emerged midges are counted and sexed at least once, but preferably twice a day (early morning and late afternoon).
Biološka pomembnost odziva VTG na zaviranje estrogena/aromataze je bila dokazana in obsežno dokumentirana (8) (9).
After identification, the midges of the 1st generation are carefully removed from the vessels and transferred to a breeding cage.
Vendar je mogoče, da lahko na nastajanje VTG pri samicah vplivajo tudi splošna strupenost in neendokrini strupeni načini delovanja.
Midges of the 2nd generation are removed and killed after identification.
Za redno uporabo za kvantifikacijo VTG v krvi, jetrih, celotnem telesu ali odvzetih vzorcih homogenata glave/repa posameznih rib je bilo uspešno razvitih in standardiziranih več merilnih metod.
Any egg ropes deposited in the test vessels of the 1st generation should be collected individually and transferred with at least 2,5 ml native water to 12-well microplates (or other suitable vessels) which are covered with a lid to prevent significant evaporation.
Tako je pri cebrici, zetu in japonski medaki ter tudi pri delno validirani vrsti črnoglavega pisanca;
The number of dead larvae and visible pupae that have failed to emerge should also be recorded.
na voljo so metode encimskega imunskega testa (ELISA), ki so specifične za posamezne vrste in uporabljajo imunokemijo za kvantifikacijo VTG (5) (10) (11) (12) (13) (14) (15) (16).
Examples of a breeding cage, test vessel and exhauster are provided in Appendix 5.
Pri japonski medaki in cebrici je dobra povezava med VTG, izmerjenim v krvni plazmi, jetrih in vzorcih homogenata, čeprav bi lahko homogenati kazali nekoliko manjše vrednosti kot plazma (17) (18) (19).
Reproduction
V Dodatku 5 so navedeni priporočeni postopki za odvzem vzorcev za analizo VTG.
Effects on reproduction are assessed via the number of egg ropes produced by the 1st generation of midges and the fertility of these egg ropes.
Sprememba fenotipskega razmerja med spoloma (deleži po spolu) so končna točka, ki izraža spremembo spola.
Once per day the egg ropes are collected from the crystallising dish that is placed in each breeding container.
Načeloma lahko estrogeni, antiestrogeni, androgeni, antiandrogeni in kemikalije, ki zavirajo steroidogenezo, vplivajo na razmerje med spoloma pri ribah, ki se razvijajo (20).
The egg ropes should be collected and transferred with at least 2,5 ml native water to a 12-wells microplate (one egg rope in each well) or other suitable vessels, which are covered with a lid to prevent significant evaporation.
Dokazano je bilo, da je sprememba spola pri cebricah delno spremenljiva (21) po izpostavljenosti kemikaliji, ki je podobna estrogenu, pri čemer je sprememba spola po izpostavljenosti kemikaliji, ki je podobna androgenu, stalna (30).
The following characteristics are documented for each egg rope:
Spol se določi kot ženski, moški, dvospolni (v eni spolni žlezi so oociti in spermatogene celice) ali nediferenciran ter se pri posameznih ribah določi s histološkim pregledom spolnih žlez.
day of production, size (normal, i.e. 1,0 ± 0,3 cm or small; typically ≤ 0,5 cm), and structure (normal = banana-form with spiralled egg string or abnormal, e.g. unspiralled egg string) and fertility (fertile or infertile).
Smernice so navedene v Dodatku 7 in Smernici OECD za določitev histopatologije, povezane z endokrinim sistemom, spolnih žlez pri ribah (22).
Over the course of six days after it was produced the fertility of an egg rope is assessed.
Genetski spol se pregleda prek genetskih označevalcev, kadar obstajajo pri dani vrsti rib.
An egg rope is considered fertile when at least one third of the eggs hatch.
Pri japonski medaki se lahko ženski geni XX ali moški geni XY določijo z verižno reakcijo s polimerazo (PCR) ali z analizo na kromosom Y vezanega gena z domeno DM (prisotnost ali odsotnost domene DM), kot je opisano v (23) (24).
The total number of females added to the breeding cage is used to calculate the number of egg ropes per female and the number of fertile egg ropes per female.
Za zeta obstaja enakovredna metoda PCR za določitev genetskega spola, ki je opisana v Dodatku 10.
If required, the number of eggs in an egg rope can be estimated non-destructively by using the ring count method (detailed in 32 and 33).
Če se lahko posamezni genetski spol poveže s fenotipičnim spolom, se vrednost preskusa izboljša, pri čemer je zato treba genetski spol določiti pri vrstah z dokumentiranimi označevalci genetskega spola.
Analytical measurements
Glavni endokrini končni točki, tj. VTG in razmerje med spoloma, lahko skupaj dokažeta endokrini način delovanja (MOA) kemikalije (tabela 1).
Concentration of the test chemical
Razmerje med spoloma je biomarker, pomemben za populacijo (25) (26), za nekatere dobro opredeljene načine delovanja pa se lahko rezultati preskusa spolnega razvoja rib uporabijo za namene ocene nevarnosti in tveganja, če regulativna agencija meni, da je to ustrezno.
As a minimum, samples of the overlying water, pore water and the sediment should be analysed at the start of exposure (in case of water spiking preferably one hour after application) and at the end of the test, at the highest concentration and a lower one.
Ti načini delovanja so zdaj estrogeni, androgeni in inhibitorji steroidogeneze.
This applies to vessels from both generations.
Odziv endokrinih končnih točk na različne načine delovanja kemikalij:
From the crystallising dishes in the breeding cage only the overlying water is analysed, since this is what the egg ropes come into contact with (for the spiked sediment design an analytical confirmation of the sediment concentration may be considered).
↑= povečevanje, ↓= zmanjševanje, – = se ne proučuje
Further measurements of sediment, pore water or overlying water during the test may be conducted if deemed necessary.
Viri
These determinations of test chemical concentration inform on the behaviour/partitioning of the test chemical in the water-sediment system.
šibek agonist estrogena
Sampling of sediment and pore water at the start and during the test (see paragraph 39) requires additional test vessels to perform analytical determinations.
↑ ♀ ali ↑ nedif.
Measurements in sediment in the spiked water design might not be necessary if the partitioning of the test chemical between water and sediment has been clearly determined in a water/sediment study under comparable conditions (e.g. sediment to water ratio, type of application, organic carbon content of sediment), or if measured concentrations in the overlying water are shown to remain within 80 to 120 % of the nominal or measured initial concentrations..
močan agonist estrogena
When intermediate measurements are made (e.g. at day 7 and/or 14) and if the analysis needs large samples which cannot be taken from test vessels without influencing the test system, analytical determinations should be performed on samples from additional test vessels treated in the same way (including the presence of test organisms) but not used for biological observations.
↑ ♀ ali ↑ nedif., ne ♂
Centrifugation at e.g. 10000 g at 4 °C for 30 min is the recommended procedure to isolate interstitial (= pore) water.
antagonist estrogena
However, if the test chemical is demonstrated not to adsorb to filters, filtration may also be acceptable.
↓ ♀, ↑ nedif.
In some cases it might not be possible to analyse concentrations in the pore water as the sample volume may be too small.
agonist androgena
Physical-chemical parameters
↑ ♂, ne ♀
pH, dissolved oxygen in the test water and temperature of the water in the test vessels and crystallising dishes should be measured in an appropriate manner (see paragraph 10).
antagonist androgena
Hardness and ammonia should be measured in the controls and in one test vessel and crystallising dish at the highest concentration at the start and the end of the test.
↑ dvospolni
The purpose of this life-cycle test is to determine the effect of the test chemical on the reproduction and, for two generations, the development rate and the total number of fully emerged and alive male and female midges.
inhibitor aromataze
For the emergence ratio data of males and females should be pooled.
Preskus spolnega razvoja rib ne zajema razmnoževalne življenjske faze rib, zato je treba kemikalije, ki domnevno vplivajo na razmnoževanje pri nižjih koncentracijah kot na spolni razvoj, proučiti s preskusom, ki zajema razmnoževanje.
If there are no statistically significant differences between the sensitivities in the development rate of the separate sexes, male and female results may be pooled for statistical analysis.
Opredelitve pojmov za namen te preskusne metode so navedene v Dodatku 1.
Effect concentrations expressed as concentrations in the overlaying water (for spiked water) or in the sediment (for spiked sediment), are usually calculated based on measured concentrations at the beginning of the exposure (see paragraph 38).
Cilj preskusa spolnega razvoja rib in vivo je določitev kemikalij z androgenimi in estrogenimi lastnostmi ter antiandrogenimi lastnostmi, antiestrogenimi lastnostmi in lastnostmi, ki zavirajo steroidogenezo.
Therefore, for spiked water, the concentrations typically measured at the beginning of the exposure in the overlying water of the vessels for both generations and those of the crystallising dishes are averaged for each treatment.
Fazi validacije preskusa spolnega razvoja rib (1 in 2) sta zajeli estrogene in androgene kemikalije ter kemikalije, ki zavirajo steroidogenezo.
For spiked sediment, the concentrations typically measured at the beginning of the exposure in the vessels for both generations (and optionally those of the crystallising dishes) are averaged for each treatment.
Učinki na antagoniste estrogena in androgena v preskusu spolnega razvoja rib so navedeni v tabeli 1, vendar so ti načini delovanja zdaj manj dokumentirani.
To compute a point estimate, i.e. an ECx, the per-vessel and per-breeding cage statistics may be used as true replicates.
V preskusu so ribe od novo oplojene ikre do konca spolne diferenciacije izpostavljene vsaj trem koncentracijam preskusne kemikalije, raztopljene v vodi.
In calculating a confidence interval for any ECx the variability among vessels should be taken into account, or it should be shown that this variability is so small that it can be ignored.
Preskusni pogoji morajo biti pretočni, razen če niso mogoči zaradi razpoložljivosti ali narave (npr. omejena topnost) preskusne kemikalije.
When the model is fitted by Least Squares, a transformation should be applied to the per-vessel statistics in order to improve the homogeneity of variance.
Preskus se začne z vnosom novo oplojenih iker (pred brazdanjem blastodiska) v preskusne komore.
However, ECx values should be calculated after the response is transformed back to the original value (31).
Vnos posameznih vrst v komore je opisan v odstavku 27.
When the statistical analysis aims at determining the NOEC by hypothesis testing, the variability among vessels needs to be taken into account, which is guaranteed by using ANOVA methods (e.g. Williams' and Dunnett's test procedures).
Pri validiranih vrstah, tj. japonski medaki, zetu in cebrici, se preskus konča 60 dni po izvalitvi.
Williams' test would be appropriate when a monotonic dose-response is expected in theory and Dunnett's test would be appropriate where the monotonicity hypothesis does not hold.
Po koncu preskusa se vse ribe humano evtanazirajo.
Alternatively, more robust tests (27) can be appropriate in situations where there are violations of the usual ANOVA assumptions (31).
Za analizo VTG se odvzamejo biološki vzorci (krvna plazma, jetra ali homogenat glave/repa) posameznih rib, preostali del pa se fiksira za histološko oceno spolnih žlez, da se določi fenotipični spol;
Emergence ratio
neobvezno se lahko opravi histopatologija (npr. določanje faze spolnih žlez, stopnja dvospolnosti).
Emergence ratios are quantal data, and can be analysed by the Cochran-Armitage test applied in a step-down manner where a monotonic dose-response is expected and these data are consistent with this expectation.
Pri vrstah, ki imajo ustrezne označevalce, se odvzame biološki vzorec (predrepna ali hrbtna plavut) za določitev genetskega spola (dodatka 9 in 10).
If not, a Fisher's exact or Mantel-Haentzal test with Bonferroni-Holm adjusted p-values can be used.
Na voljo morajo biti rezultati preskusa akutne strupenosti ali drugega kratkoročnega preskusa strupenosti [npr. preskusna metoda C.14 (34) in OECD TG 210 (1)], po možnosti izvedenega z vrstami, izbranimi za ta preskus.
If there is evidence of greater variability between replicates within the same concentration than a binomial distribution would indicate (often referenced to as “extra-binomial” variation), then a robust Cochran-Armitage or Fisher exact test such as proposed in (27), should be used.
To pomeni, da sta znana topnost preskusne kemikalije v vodi in parni tlak preskusne kemikalije ter da je na voljo zanesljiva analitska metoda za kvantifikacijo kemikalije v preskusnih komorah z znano in izpričano točnostjo ter mejo zaznavnosti.
The sum of live midges (males plus females) emerged per vessel, ne, is determined and divided by the number of larvae introduced, na:
Druge uporabne informacije so strukturna formula, čistost kemikalije, stabilnost v vodi ali na svetlobi, Pow in rezultati preskusa za lahko biološko razgradljivost (metoda C.4) (35).
ER emergence ratio
Merila za sprejemljivost preskusa
number of live midges emerged per vessel
koncentracija raztopljenega kisika mora biti ves čas preskusa vsaj 60 % nasičenosti z zrakom (ASV),
number of larvae introduced per vessel (normally 20)
razlika v temperaturi vode v različnih preskusnih komorah med obdobjem izpostavljenosti nikoli ne sme biti večja od ± 1,5 °C in jo je treba vzdrževati znotraj temperaturnih razponov, ki so določeni za preskusno vrsto (Dodatek 2),
When ne is larger than na (i.e. when unintentionally more than the foreseen number of larvae where introduced) na should be made equal to ne.
na voljo mora biti validirana metoda za analizo kemikalije za izpostavljenost z mejo zaznavnosti, ki je znatno nižja od najnižje nominalne koncentracije, zbrati pa je treba dokaze, da so bile koncentracije preskusne kemikalije v raztopini zadovoljivo ohranjene v razponu ± 20 % srednjih izmerjenih vrednosti,
An alternative approach that is most appropriate for large sample sizes, when there is extra binomial variance, is to treat the emergence ratio as a continuous response and use procedures consistent with these ER data.
število vseh preživelih oplojenih iker v kontrolah in, kadar je primerno, v kontrolah s topilom mora biti večje ali enako mejnim vrednostim iz Dodatka 2,
A large sample size is defined here as the number emerged and the number not emerging both exceeding five, on a per replicate (vessel) basis.
merila sprejemljivosti, povezana z rastjo in lastnostmi spola ob koncu preskusa, temeljijo na podatkih iz kontrolnih skupin (združene kontrole s topilom in vodo, če pa se zelo razlikujejo, samo s topilom):
To apply ANOVA methods, values of ER should first be transformed by the arcsin-sqrt transformation or Tukey-Freeman transformation to obtain an approximate normal distribution and to equalise variances.
Japonska medaka
The Cochran-Armitage, Fisher's exact (Bonferroni), or Mantel-Haentzal tests can be applied when using the absolute frequencies.
Zet
The arcsin-sqrt transformation is applied by taking the inverse sine (sine– 1) of the square root of ER.
Rast
For emergence ratios, ECx-values are calculated using regression analysis (e.g. probit, logit or Weibull models (28)).
Mokra teža ribe (osušene s pivnikom)
If regression analysis fails (e.g. when there are less than two partial responses), other non-parametric methods such as moving average or simple interpolation can be used.
> 75 mg
Development rate
Dolžina (standardna dolžina)
Mean development time represents the mean time span between the introduction of larvae (day 0 of the test) and the emergence of the experimental cohort of midges (for calculation of the true development time, the age of larvae at the time of introduction should be considered).
> 20 mm
The development rate (unit: 1/day) is the reciprocal of the development time and represents that portion of larval development which takes place per day.
> 14 mm
Development rate is preferred for the evaluation of these sediment toxicity studies as its variance is lower, and it is more homogeneous and closer to a normal distribution compared to the development time.
razmerje med spoloma (% samcev ali samic)
Hence, more powerful parametric test procedures may be used with development rate unlike development time.
Če se uporabi topilo, ne sme imeti statistično značilnega učinka na preživetje in ne sme povzročati učinkov endokrinih motilcev ali drugih škodljivih učinkov na zgodnje življenjske faze, kar se pokaže s kontrolo s topilom.