natgillin/translations-raw huggingface dataset:
The choice of solvent will be determined by the chemical properties of the substance or mixture.
E – izpuščaji na spodnji čeljusti, blizu ust, so običajno majhni in pogosto v parih.
The OECD guidance document recommends a maximum of 100 μl/l, which should be observed.
Pri kontrolnih ali tretiranih samcih in tretiranih samicah se razlikujejo.
However a recent review (24) highlighted additional concerns when using solvents for endocrine activity testing.
F – izpuščaji, locirani ventralno od E. Pogosto so majhni in v parih.
Therefore it is recommended that the solvent concentration, if necessary, is minimised wherever technically feasible (dependent on the physical-chemical properties of the test chemical).
Prisotni so pri kontrolnih samcih in samicah, izpostavljenih androgenom.
A flow-through test system will be used.
Predloga za izpuščaje:
Such a system continually dispenses and dilutes a stock solution of the test chemical (e.g. metering pump, proportional diluter, saturator system) in order to deliver a series of concentrations to the test chambers.
Številska ocena
The flow rates of stock solutions and dilution water should be checked at intervals, preferably daily, during the test and should not vary by more than 10 % throughout the test.
Datum
Care should be taken to avoid the use of low-grade plastic tubing or other materials that may contain biologically active chemicals.
1- prisoten
When selecting the material for the flow-through system, possible adsorption of the test chemical to this material should be considered.
Skupna ocena
Test fish should be selected from a laboratory population, preferably from a single stock, which has been acclimated for at least two weeks prior to the test under conditions of water quality and illumination similar to those used in the test.
2- povečan
It is important that the loading rate and stocking density (for definitions, see Appendix 1) be appropriate for the test species used (see Appendix 2).
3- izrazit
mortalities of greater than 10 % of population in seven days: reject the entire batch;
Dodatek 5B
mortalities of between 5 % and 10 % of population: acclimation for seven additional days; if more than 5 % mortality during second seven days, reject the entire batch;
OCENA SEKUNDARNIH SPOLNIH ZNAČILNOSTI PRI MEDAKI ZA ZAZNAVANJE NEKATERIH KEMIKALIJ Z ENDOKRINIM DELOVANJEM
mortalities of less than 5 % of population in seven days: accept the batch.
V nadaljevanju je naveden opis meritve bradavičastih podaljškov [1], ki so sekundarna spolna značilnost pri medaki (Oryzias latipes).
Fish should not receive treatment for disease during the acclimation period, in the pre-exposure period, or during the exposure period.
Po odstranitvi jeter (Dodatek 6) se telo postavi v konično epruveto, ki vsebuje približno 10 ml 10-odstotnega nevtralno pufranega formalina (glava navzgor in rep navzdol).
Pre-exposure and selection of fish
Če so spolne žleze fiksirane v raztopini, ki ni 10-odstotni nevtralno pufran formalin, se z rezilom naredi prečni rez čez telo med anteriornim območjem predrepne plavuti in anusom, pri čemer je treba paziti, da se ne poškodujejo gonopora in spolne žleze (slika 3).
The one to two-week pre-exposure period is recommended with animals placed in vessels similar to the actual test.
Kranialna stran telesa ribe se vstavi v fiksirno raztopino, da se ohranijo spolne žleze, repna stran telesa ribe pa se vstavi v 10-odstotni nevtralno pufran formalin, kot je opisano zgoraj.
Fish should be fed ad libitum throughout the holding period and during the exposure phase.
Ko se telo ribe vstavi v 10-odstotni nevtralno pufran formalin, se anteriorno območje predrepne plavuti prime s pinceto in za približno 30 sekund prepogne, da je predrepna plavut odprta.
The exposure phase is started with sexually dimorphic adult fish from a laboratory supply of reproductively mature animals (e.g. with clear secondary sexual characteristics visible as far as fathead minnow and medaka are concerned), and actively spawning.
Pri prijemu predrepne plavuti s pinceto se nekaj plavutnic v anteriornem območju previdno prime, da se bradavičasti podaljški ne opraskajo.
For general guidance only (and not to be considered in isolation from observing the actual reproductive status of a given batch of fish), fathead minnows should be approximately 20 (± 2) weeks of age, assuming they have been cultured at 25 ± 2 °C throughout their lifespan.
Ko je predrepna plavut približno 30 sekund odprta, se telo ribe shrani v 10-odstotnem nevtralno pufranem formalinu pri sobni temperaturi do meritve bradavičastih podaljškov (meritev je treba izvesti po vsaj 24-urni fiksaciji).
Japanese medaka should be approximately 16 (± 2) weeks of age, assuming they have been cultured at 25 ± 2 °C throughout their lifespan.
Merjenje
Zebrafish should be approximately 16 (± 2) weeks of age, assuming they have been cultured at 26 ± 2 °C throughout their lifespan.
Po vsaj 24-urni fiksaciji telesa ribe v 10-odstotnem nevtralnem pufranem formalinu se telo ribe vzame iz konične epruvete in formalin obriše s filtrirnim papirjem (ali papirnato brisačo).
Egg production should be assessed daily during the pre-exposure phase.
Riba se obrne s trebušno stranjo navzgor.
It is recommended that spawning be observed in all replicate tanks prior to inclusion in the exposure phase of the assay.
Nato se predrepna plavut previdno odreže z majhnimi secirnimi škarjami (predrepna plavut se po možnosti odreže z nekaj radii (pterygiophore)).
Quantitative guidance on desirable daily egg production cannot be provided at this stage, but it is relatively common to observe average spawns of > 10 eggs/female/day for each species.
Anteriorni del odrezane predrepne plavuti se prime s pinceto in položi na objektno stekelce z nekaj kapljicami vode.
A randomised block design according to egg production output should be used to allocate replicates to the various experimental levels to ensure balanced distribution of replicates.
Nato se predrepna plavut prekrije s krovnim stekelcem.
TEST DESIGN
Med prijemanjem predrepne plavuti s pinceto je treba paziti, da se bradavičasti podaljški ne opraskajo.
Three concentrations of the test chemical, one control (water) and, if needed, one solvent control are used.
S števcem se pod biološkim mikroskopom (pokončni mikroskop ali obrnjeni mikroskop) prešteje število ploščic z bradavičastimi podaljški.
The data may be analysed in order to determine statistically significant differences between treatment and control responses.
Bradavičasti podaljški se prepoznajo, če je na posteriornem robu ploščice vidna majhna tvorba podaljškov.
These analyses will inform whether further longer term testing for adverse effects (namely, survival, development, growth and reproduction) is required for the chemical, rather than for use in risk assessment (25).
Na delovni list se zapiše število ploščic z bradavičastimi podaljški na vsaki plavutnici (npr. prva plavutnica: 0, druga plavutnica: 10, tretja plavutnica: 12 itd.), vsota teh števil po posameznih ribah pa se vnese v Excelovo preglednico.
For zebrafish, on day 21 of the experiment, males and females from each treatment level (5 males and 5 females in each of the two replicates) and from the control(s) are sampled for the measurement of vitellogenin.
Po potrebi se predrepna plavut fotografira in na fotografiji prešteje število ploščic z bradavičastimi podaljški.
For medaka, on day 21 of the experiment, males and females from each treatment level (3 males and 3 females in each of the four replicates) and from the control(s) are sampled for the measurement of vitellogenin and secondary sex characteristics.
Po meritvi se predrepna plavut vstavi v konično epruveto, opisano v (1), in shrani.
For fathead minnow, on day 21 of exposure, males and females (2 males and 4 females in each of the four replicates) and from the control(s) are sampled for the measurement of vitellogenin and secondary sex characteristics.
Slika 1
Quantitative assessment of fecundity is required, and gonadal tissues should be fixed in whole or dissected for potential histopathological evaluation, if required.
Diagram, na katerem so prikazane razlike v obliki in velikosti predrepne plavuti glede na spol.
Selection of test concentrations
A: samec, B: samica.
For the purposes of this test, the highest test concentration should be set by the maximum tolerated concentration (MTC) determined from a range finder or from other toxicity data, or 10 mg/l, or the maximum solubility in water, whichever is lowest.
Slika 2
The MTC is defined as the highest test concentration of the chemical which results in less than 10 % mortality.
Slika 3
Using this approach assumes that there are existing empirical acute toxicity data or other toxicity data from which the MTC can be estimated.
Fotografija telesa ribe, na kateri je prikazana stran reza, če so spolne žleze fiksirane v raztopini za fiksiranje, ki ni 10-odstotni nevtralno pufran formalin.
Estimating the MTC can be inexact and typically requires some professional judgment.
V tem primeru se preostalo telo z rezilom (rdeča) prereže med anteriornim delom predrepne plavuti in anusom, sprednji del ribe se vstavi v raztopino za fiksiranje spolnih žlez, zadnji del ribe pa se vstavi v 10-odstotni nevtralno pufran formalin.
Three test concentrations, spaced by a constant factor not exceeding 10, and a dilution-water control (and solvent control if necessary) are required.
Bradavičasti podaljški se običajno pojavijo le pri odraslih samcih na plavutnicah, in sicer od druge do sedme ali osme s posteriornega konca predrepne plavuti (sliki 1 in 2).
A range of spacing factors between 3,2 and 10 is recommended.
Vendar se podaljški redko pojavijo na prvi plavutnici s posteriornega konca predrepne plavuti.
Selection and weighing of test fish
Ta standardni operativni postopek zajema meritev podaljškov na prvi plavutnici (v tem standardnem operativnem postopku se številka plavutnice nanaša na zaporedje od posteriornega konca predrepne plavuti).
It is important to minimise variation in weight of the fish at the beginning of the assay.
PRIPOROČENI POSTOPKI ZA ODVZEM VZORCEV ZA ANALIZO VITELOGENINA
Suitable size ranges for the different species recommended for use in this test are given in Appendix 2.
Preprečiti je treba navzkrižno kontaminacijo med vzorci VTG samcev in samic.
For the whole batch of fish used in the test, the range in individual weights for male and female fish at the start of the test should be kept, if possible, within ± 20 % of the arithmetic mean weight of the same sex.
Postopek 1A:
It is recommended to weigh a subsample of the fish stock before the test in order to estimate the mean weight.
Črnoglavi pisanec, odvzem krvi iz repne vene/arterije
The test duration is 21 days, following a pre-exposure period.
Po anesteziji se repno steblo delno prereže z rezilom skalpela, pri čemer se kri odvzame iz repne vene/arterije s heparinizirano mikrohematokritsko kapilarno cevko.
The recommended pre-exposure period is one to two weeks.
Ko je kri odvzeta, se plazma hitro izolira s 3-minutnim centrifugiranjem pri 15000 g (ali z 10-minutnim centrifugiranjem pri 15000 g in 4 °C).
Fish should be fed ad libitum with an appropriate food (Appendix 2) at a sufficient rate to maintain body condition.
Po želji se lahko po centrifugiranju določi odstotek hematokrita.
Care should be taken to avoid microbial growth and water turbidity.
Plazemski del se nato odstrani iz mikrohematokritske cevke in shrani v centrifugalni epruveti z 0,13 enotami aprotinina (zaviralec proteaze) pri –80 °C, dokler ni mogoče določiti VTG.
As a general guidance, the daily ration may be divided into two or three equal portions for multiple feeds per day, separated by at least three hours between each feed.
Glede na velikost črnoglavega pisanca (ki je odvisna od spola) se običajno lahko odvzame od 5 do 60 mikrolitrov plazme na ribo (Jensen et al.
A single larger ration is acceptable particularly for weekends.
2001).
Food should be withheld from the fish for 12 hours prior to sampling/necropsy.
Postopek 1B:
Fish food should be evaluated for the presence of contaminants such as organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs).
Črnoglavi pisanec, odvzem krvi iz srca
Food with an elevated level of phytoestrogens that would compromise the response of the assay to known oestrogen agonist (e.g. 17β- estradiol) should be avoided.
Namesto tega se lahko kri odvzame tudi s srčno punkcijo s heparinizirano brizgo (1000 enot heparina na ml).
Uneaten food and faecal material should be removed from the test vessels at least twice weekly, e.g. by carefully cleaning the bottom of each tank using a siphon.
Kri se prenese v Eppendorfove epruvete (shranjene na ledu) in nato centrifugira (5 minut, 7000 g, sobna temperatura).
Prior to initiation of the exposure period, proper function of the chemical delivery system should be ensured.
Plazmo je treba prenesti v čiste Eppendorfove epruvete (v alikvotih, če količina plazme to omogoča) in takoj zamrzniti pri –80 °C, dokler se ne analizira (Panter et al.
All analytical methods needed should be established, including sufficient knowledge on the chemical stability in the test system.
1998).
During the test, the concentrations of the test chemical are determined at regular intervals, as follows: the flow rates of diluent and toxicant stock solution should be checked preferably daily but as a minimum twice per week, and should not vary by more than 10 % throughout the test.
Postopek 2A:
It is recommended that the actual test chemical concentrations be measured in all vessels at the start of the test and at weekly intervals thereafter.
Japonska medaka, odstranitev jeter pri medaki
It is recommended that results be based on measured concentrations.
Odstranitev preskusne ribe iz preskusne komore
However, if 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.
Preskusno ribo je treba iz preskusne komore odstraniti z lovilno mrežico.
Samples may need to be filtered (e.g. using a 0,45 μm pore size) or centrifuged.
Paziti je treba, da preskusna riba ne pade v druge preskusne komore.
If needed, then centrifugation is the recommended procedure.
Načeloma je treba preskusne ribe odstraniti po naslednjem vrstnem redu: kontrola, kontrola s topilom (kadar je to ustrezno), najnižja koncentracija, srednja koncentracija, najvišja koncentracija in pozitivna kontrola.
However, if the test material does not adsorb to filters, filtration may also be acceptable.
Poleg tega je treba odstraniti vse samce iz ene preskusne komore, preden se odstranijo preostale samice.
During the test, dissolved oxygen, temperature, and pH should be measured in all test vessels at least once per week.
Spol posamezne preskusne ribe se določi na podlagi zunanjih spolnih značilnosti (npr. oblike predrepne plavuti).
Total hardness and alkalinity should be measured in the controls and one vessel at the highest concentration at least once per week.
Preskusna riba se vstavi v posodo za prenos in prenese na delovno postajo za odstranitev jeter.
A number of general (e.g. survival) and biological responses (e.g. VTG levels) are assessed over the course of the assay or at termination of the assay.
Preveri se, ali so oznake preskusne komore in posode za prenos točne, ter potrdi, da sta število rib, ki so bile odstranjene iz preskusne komore, in število rib, ki ostanejo v preskusni komori, skladni s pričakovanji.
The daily quantitative monitoring of fecundity is required.
Če spola ni mogoče določiti na podlagi zunanjega videza rib, se iz preskusne komore odstranijo vse ribe.
Measurement and evaluation of these endpoints and their utility are described below.
V tem primeru je treba spol določiti z opazovanjem spolnih žlez ali sekundarnih spolnih značilnosti pod stereomikroskopom.
Survival
Odstranitev jeter
Fish should be examined daily during the test period and any mortality should be recorded and the dead fish removed as soon as possible.
Preskusna riba se iz posode za prenos z lovilno mrežico prenese v raztopino za anestezijo.
Dead fish should not be replaced in either the control or treatment vessels.
Ko je preskusna riba anestezirana, se s pinceto (za splošno rabo) prenese na filtrirni papir (ali papirnato brisačo).
Sex of fish that die during the test should be determined by macroscopic evaluation of the gonads.
S pinceto se prime glava preskusne ribe, da se prepreči poškodba repa.
Behaviour and appearance
S površine preskusne ribe se s filtrirnim papirjem (ali papirnato brisačo) obriše voda.
Any abnormal behaviour (relative to controls) should be noted; this might include signs of general toxicity including hyperventilation, uncoordinated swimming, loss of equilibrium, and atypical quiescence or feeding.
Nato se z majhnimi secirnimi škarjami naredi majhen prečni rez na sredini med ventralnim delom vratu in srednjim trebušnim delom.
Additionally external abnormalities (such as haemorrhage, discoloration) should be noted.
V majhni rez se vstavijo secirne škarje, s katerimi se zareže v trebuh od točke pod škržnim plaščem do kranialne strani anusa po sredini trebuha.
Such behavioural observations may also provide useful qualitative information to inform potential future fish testing requirements.
Pod stereomikroskopom se izvedejo naslednji postopki.
Because some aspects of appearance (primarily colour) can change quickly with handling, it is important that qualitative observations be made prior to removal of animals from the test system.
Stene trebušne votline se razprejo z natančno pinceto, notranji organi pa se eksteriorizirajo.
Experience to date with fathead minnows suggests that some endocrine active chemicals may initially induce changes in the following external characteristics: body colour (light or dark), coloration patterns (presence of vertical bands), and body shape (head and pectoral region).
Po potrebi je sprejemljiva tudi eksteriorizacija notranjih organov z odstranitvijo ene strani stene trebušne votline.
Daily quantitative observations of spawning should be recorded on a replicate basis.
Paziti je treba, da se žolčnik ne poškoduje.
Egg production should be recorded as the number of eggs/surviving female/day on a replicate basis.
Na enak način se prime požiralnik in prebavni trakt odreže od jeter.
Eggs will be removed daily from the test chambers.
Paziti je treba, da iz prebavnega trakta ne uide vsebina.
Spawning substrates should be placed in the test chamber for the fathead minnow and zebrafish to enable fish to spawn in normal conditions.
Odreže se repni prebavni trakt od anusa in odstrani trakt iz trebušne votline.
Appendix 4 gives further details of recommended spawning substrates for zebrafish (Appendix 4A) and fathead minnow (Appendix 4B).
Z obrobja jeter se odrežeta masa maščobe in drugo tkivo.
It is not considered necessary to provide spawning substrate for medaka.
Paziti je treba, da se jetra ne opraskajo.
Observation of secondary sex characteristics
Po potrebi se s površine jeter z natančno pinceto odstranita vsa preostala maščoba in nepripadajoče tkivo (npr. trebušna mrena).
Notably, chemicals with certain modes of action may cause abnormal occurrence of secondary sex characteristic in animals of the opposite sex; for example, androgen receptor agonists, such as trenbolone, methyltestosterone and dihydrotestosterone, can cause female fathead minnows to develop pronounced nuptial tubercles or female medaka to develop papillary processes (11, 20, 21).
Vrednost se zapiše na delovni list (na 0,1 mg natančno).
Such gross morphological observations may provide useful qualitative and quantitative information to inform potential future fish testing requirements.
Pokrov mikroepruvete z jetri se zapre.
Recommended procedures for the evaluation of secondary sex characteristics in fathead minnow and medaka are available from Appendix 5A and Appendix 5B, respectively.
Po odstranitvi enih jeter se instrumenti za seciranje očistijo ali nadomestijo s čistimi.
Vitellogenin (VTG)
Jetra se iz vseh rib v posodi za prenos odstranijo, kot je opisano zgoraj.
Depending upon the size of the fish, collectable blood volumes generally range from 5 to 60 μl per individual for fathead minnows and 5-15 μl per individual for zebrafish.
Če se jetra donirajo za predhodno tretiranje kmalu po odstranitvi, se vzorci prenesejo na naslednjo delovno postajo na hladilnem (ali zamrzovalnem stojalu).
Plasma is separated from the blood via centrifugation, and stored with protease inhibitors at – 80 °C, until analysed for VTG.
Po odstranitvi jeter je telo ribe na voljo za histologijo spolnih žlez in meritev sekundarnih spolnih značilnosti.
The measurement of VTG should be based upon a validated homologous ELISA method, using homologous VTG standard and homologous antibodies.
Vzorci jeter, odvzeti pri preskusnih ribah, se shranijo pri ≤ –70 °C, če se ne uporabijo za predhodno tretiranje kmalu po odstranitvi.
It is recommended to use a method capable to detect VTG levels as low as few ng/ml plasma (or ng/mg tissue), which is the background level in unexposed male fish.
Rez se naredi s škarjami neposredno anteriorno od prsne plavuti.
For each ELISA method, a test for matrix effect (effect of sample dilution) should be run to determine the minimum sample dilution factor.
Trebušne stene se razširijo s prijemalkami, da se izpostavijo jetra in drugi notranji organi.
Each ELISA plate used for VTG assays should include the following quality control samples: at least 6 calibration standards covering the range of expected VTG concentrations, and at least one non-specific binding assay blank (analysed in duplicate).
(Namesto tega se lahko trebušne stene pripnejo s strani.)
At least two aliquots (well-duplicates) of each sample dilution will be analysed.
Jetra se secirajo in odstranijo s prijemalkami.
The correlation coefficient (R2) for calibration curves should be greater than 0,99.
Črevesje se s prijemalkami nežno odmakne.
However, a high correlation is not sufficient to guarantee adequate prediction of concentration in all ranges.
Slika 6
In addition to having a sufficiently high correlation for the calibration curve, the concentration of each standard, as calculated from the calibration curve, should all fall between 70 and 120 % of its nominal concentration.
Oba konca črevesja in vse pritrditve mezenterija se odrežejo s škarjami.
If the nominal concentrations trend away from the calibration regression line (e.g. at lower concentrations), it may be necessary to split the calibration curve into low and high ranges or to use a nonlinear model to adequately fit the absorbance data.
Slika 7 (samica)
If the curve is split, both line segments should have R2 > 0,99.
Postopek za samico je enak.
The limit of detection (LOD) is defined as the concentration of the lowest analytical standard, and limit of quantitation (LOQ) is defined as the concentration of the lowest analytical standard multiplied by the lowest dilution factor.
Slika 8
On each day that VTG assays are performed, a fortification sample made using an inter-assay reference standard will be analysed (Appendix 7).
Končan postopek.
The ratio of the expected concentration to the measured concentration will be reported along with the results from each set of assays performed on that day.
Postopek 2B:
Evaluation of gonadal histopathology
Japonska medaka (Oryzias latipes), predhodno tretiranje jeter za analizo vitelogenina
In this respect, gonads are fixed either whole body or dissected.
Če se uporabi homogenizacijski pufer iz sistema EnBio ELISA, se raztopina odtaja pri sobni temperaturi, nato pa se steklenica ohladi z zdrobljenim ledom.
When histopathology is required, specific endocrine-related responses on the gonads will be looked for in the assessment of the endocrine activity of the test chemical.
Količina homogenizacijskega pufra za jetra se izračuna na podlagi njihove teže (doda se 50 μl homogenizacijskega pufra na miligram teže jeter).
These diagnostic responses essentially include the presence of testicular oocytes, Leydig cell hyperplasia, decreased yolk formation, increased spermatogonia and perifollicular hyperplasia.
Če jetra tehtajo na primer 4,5 mg, je količina homogenizacijskega pufra za jetra 225 μl.