natgillin/translations-raw huggingface dataset:
Fish should not receive treatment for disease during the acclimation period, in the pre-exposure period, or during the exposure period.
Broj i veličina izraslina u spolno zrele debeloglave gavčice i papilarnih procesa u medake mogu se kvantificirati izravno ili praktičnije iz očuvanih uzoraka.
Pre-exposure and selection of fish
Preporučeni postupci za procjenu sekundarnih spolnih obilježja u debeloglave gavčice i medake nalaze se u Dodatku 5.A odnosno Dodatku 5.B.
The one to two-week pre-exposure period is recommended with animals placed in vessels similar to the actual test.
Vitelogenin (VTG)
Fish should be fed ad libitum throughout the holding period and during the exposure phase.
Krv se uzima iz repne arterije/vene s pomoću heparinizirane mikrohematokritske kapilarne cjevčice ili punkcijom srca s pomoću šprice.
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.
Ovisno o veličini ribe, količina krvi koja se uzima u pravilu se kreće od pet do 60 μl po jednoj debeloglavoj gavčici, odnosno od pet do 15 μl po jednoj zebrici.
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.
Plazma se odvaja od krvi centrifugiranjem te pohranjuje s inhibitorima proteaze na – 80 °C do analize VTG-a.
Japanese medaka should be approximately 16 (± 2) weeks of age, assuming they have been cultured at 25 ± 2 °C throughout their lifespan.
Druga je mogućnost upotreba jetre medake, odnosno homogenata s glave/repa zebrice kao izvora tkiva za određivanje VTG-a (Dodatak 6.).
Zebrafish should be approximately 16 (± 2) weeks of age, assuming they have been cultured at 26 ± 2 °C throughout their lifespan.
Mjerenje VTG-a trebalo bi se temeljiti na validiranoj odgovarajućoj metodi ELISA s pomoću odgovarajućeg standarda VTG-a i odgovarajućih antitijela.
Egg production should be assessed daily during the pre-exposure phase.
Preporučuje se primjena metode s pomoću koje se mogu otkriti niske razine VTG-a od nekoliko ng/ml plazme (ili ng/mg tkiva), što je osnovna razina u neizloženih mužjaka riba.
It is recommended that spawning be observed in all replicate tanks prior to inclusion in the exposure phase of the assay.
Kontrola kvalitete analize VTG-a obavljat će se s pomoću standarda, slijepih proba i najmanje dviju identičnih analiza.
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.
Za svaku metodu ELISA potrebno je provesti ispitivanje za učinak matrice (učinak razrjeđivanja uzorka) radi utvrđivanja minimalnog faktora razrjeđivanja uzorka.
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.
Analizirat će se najmanje dva alikvota (dvostruka bunarića) svakog razrjeđivanja uzorka.
TEST DESIGN
Dvostruke bunariće koji se razlikuju više od 20 % potrebno je ponovno analizirati.
Three concentrations of the test chemical, one control (water) and, if needed, one solvent control are used.
Koeficijent korelacije (R2) za kalibracijske krivulje trebao bi biti veći od 0,99.
The data may be analysed in order to determine statistically significant differences between treatment and control responses.
Međutim, visoka korelacija nije dostatna za jamstvo odgovarajućeg predviđanja koncentracije u svim rasponima.
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).
Osim što ima dovoljno visoku korelaciju za kalibracijsku krivulju, koncentracija svakog standarda prema izračunu iz kalibracijske krivulje trebala bi se kretati od 70 do 120 % nazivne koncentracije.
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.
U slučaju udaljavanja nazivnih koncentracija od linije regresije kalibracije (primjerice pri nižim koncentracijama) može se pojaviti potreba za razdvajanjem kalibracijske krivulje u niske i visoke raspone ili potreba za primjenom nelinearnog modela za odgovarajuće prilagođavanje podataka o apsorbanciji.
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.
U slučaju razdvajanja krivulje, oba segmenta linije trebala bi imati R2 > 0,99.
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.
Granica otkrivanja (eng. limit of detection, LOD) definira se kao koncentracija najnižeg analitičkog standarda, dok se granica kvantitacije (eng. limit of quantitation, LOQ) definira kao koncentracija najnižeg analitičkog standarda pomnožena s najnižim faktorom razrjeđivanja.
Quantitative assessment of fecundity is required, and gonadal tissues should be fixed in whole or dissected for potential histopathological evaluation, if required.
Svakog dana provođenja testova VTG-a analizirat će se uzorak za utvrđivanje izrađen s pomoću referentnog standarda dobivenog ponavljanjem testa (inter-assay) (Dodatak 7.).
Selection of test concentrations
Omjer očekivane i izmjerene koncentracije bilježit će se uz rezultate svakog od niza testova provedenih tog dana.
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.
Procjena histopatološke pretrage gonada
The MTC is defined as the highest test concentration of the chemical which results in less than 10 % mortality.
Regulatorna tijela mogu zahtijevati histopatološku pretragu gonada radi proučavanja ciljnog organa na osi HPG nakon izlaganja kemikaliji.
Using this approach assumes that there are existing empirical acute toxicity data or other toxicity data from which the MTC can be estimated.
U tu se svrhu gonade fiksiraju u cjelini ili secirane.
Estimating the MTC can be inexact and typically requires some professional judgment.
Ako je potrebna histopatološka pretraga, pri procjeni endokrinog djelovanja ispitivane kemikalije na gonadama se promatraju specifične reakcije povezane s endokrinim sustavom.
Three test concentrations, spaced by a constant factor not exceeding 10, and a dilution-water control (and solvent control if necessary) are required.
Te dijagnostičke reakcije uglavnom uključuju prisutnost oocita u testisima, hiperplaziju Leydigovih stanica, smanjeno stvaranje žumanjka, povećane spermatogonije i perifolikularnu hiperplaziju.
A range of spacing factors between 3,2 and 10 is recommended.
Druge lezije gonada kao što su atrezija oocita, degenerativne promjene testisa i promjene stadija mogu imati različite uzroke.
Selection and weighing of test fish
U smjernicama o histopatološkim pretragama gonada riba navode se postupci koji se primjenjuju za seciranje, fiksiranje, rezanje i histopatološku procjenu spolnih žlijezda (22.).
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.
U slučaju upotrebe kontrole s otapalom potrebno je za svaku krajnju točku provesti odgovarajući statistički test između kontrola s vodom za razrjeđivanje i kontrola s otapalom.
It is recommended to weigh a subsample of the fish stock before the test in order to estimate the mean weight.
Smjernice o načinu obrade podataka za kontrole s vodom za razrjeđivanje i otapalom u naknadnoj statističkoj analizi nalaze se u dokumentu OECD, 2006c (28.).
The test duration is 21 days, following a pre-exposure period.
Sve podatke o biološkim reakcijama potrebno je analizirati i o njima izvijestiti zasebno prema spolu.
The recommended pre-exposure period is one to two weeks.
Ako nisu zadovoljene tražene pretpostavke za parametarske metode – nenormalna distribucija (npr.
Fish should be fed ad libitum with an appropriate food (Appendix 2) at a sufficient rate to maintain body condition.
Shapiro-Wilkov test) ili heterogena varijanca (Bartlettov ili Leveneov test), potrebno je razmotriti mogućnost da se prije provedbe ANOVA-e podaci preoblikuju radi homogenizacije varijanci ili da se provede ponderirana ANOVA.
Care should be taken to avoid microbial growth and water turbidity.
Za nemonotoničnu reakciju na dozu mogu se upotrijebiti Dunnettov test (parametarski) na višestrukim usporedbama parova ili Mann-Whitneyjev test s Bonferronijevom prilagodbom (neparametarski).
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.
Ako je reakcija na dozu približno monotonična, mogu se upotrijebiti ostali statistički testovi (primjerice, Jonckheere-Terpstraov ili Williamsov test).
A single larger ration is acceptable particularly for weekends.
Statistički dijagram nalazi se u Dodatku 8. i pomoć je pri odabiru najprikladnijeg statističkog testa.
Food should be withheld from the fish for 12 hours prior to sampling/necropsy.
Dodatne informacije nalaze se i u OECD-ovu Dokumentu o postojećim pristupima statističkoj analizi podataka o ekotoksičnosti (28.).
Fish food should be evaluated for the presence of contaminants such as organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs).
Izvješćivanje o rezultatima ispitivanja
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.
Podaci o ispitivanju trebali bi uključivati:
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.
Ustanova u kojoj se obavlja ispitivanje:
Prior to initiation of the exposure period, proper function of the chemical delivery system should be ensured.
odgovorne osobe i njihove odgovornosti tijekom ispitivanja,
All analytical methods needed should be established, including sufficient knowledge on the chemical stability in the test system.
svaki laboratorij treba biti dokazano stručan u upotrebi niza reprezentativnih kemikalija.
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.
karakterizaciju ispitivane kemikalije,
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.
fizikalno stanje i relevantna fizikalno-kemijska svojstva,
It is recommended that results be based on measured concentrations.
metodu i učestalost pripreme ispitnih koncentracija,
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.
informacije o stabilnosti i biorazgradivosti.
Samples may need to be filtered (e.g. using a 0,45 μm pore size) or centrifuged.
karakterizaciju otapala (svojstva, upotrijebljena koncentracija),
If needed, then centrifugation is the recommended procedure.
obrazloženje odabira otapala (ako nije riječ o vodi).
However, if the test material does not adsorb to filters, filtration may also be acceptable.
vrstu i soj,
During the test, dissolved oxygen, temperature, and pH should be measured in all test vessels at least once per week.
dobavljača i njegov objekt,
Total hardness and alkalinity should be measured in the controls and one vessel at the highest concentration at least once per week.
starost riba na početku ispitivanja i reproduktivni status / status mrijesta,
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.
podatke o postupku aklimatizacije životinja,
The daily quantitative monitoring of fecundity is required.
tjelesnu masu riba na početku izlaganja (iz poduzorka upotrijebljenog ribljeg jata).
Measurement and evaluation of these endpoints and their utility are described below.
primijenjeni postupak ispitivanja (vrsta ispitivanja, gustoća punjenja, gustoća nasada itd.),
Survival
metodu pripreme matičnih otopina i protok,
Fish should be examined daily during the test period and any mortality should be recorded and the dead fish removed as soon as possible.
nazivne ispitne koncentracije, tjedne izmjerene vrijednosti koncentracija ispitnih otopina i primijenjene analitičke metode, srednje izmjerene vrijednosti i standardne devijacije u ispitnim posudama te dokaze da se izmjerene vrijednosti odnose na koncentracije ispitivane kemikalije u stvarnoj otopini,
Dead fish should not be replaced in either the control or treatment vessels.
karakteristike vode za razrjeđivanje (uključujući pH-vrijednost, tvrdoću, lužnatost, temperaturu, koncentraciju otopljenog kisika, razine rezidualnog klora, ukupni organski ugljik, suspendirane krute tvari i sve ostale izmjerene vrijednosti),
Sex of fish that die during the test should be determined by macroscopic evaluation of the gonads.
kakvoću vode u ispitnim posudama: pH-vrijednost, tvrdoću, temperaturu i koncentraciju otopljenog kisika,
Behaviour and appearance
precizne informacije o hranjenju (primjerice vrsti/vrstama hrane, porijeklu, količini i učestalosti hranjenja te analizu relevantnih kontaminanata ako postoji (primjerice poliklorirani bifenili, policiklički aromatski ugljikovodici i organoklorni pesticidi).
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.
dokaze da su kontrole u skladu s kriterijima prihvatljivosti ispitivanja,
Such behavioural observations may also provide useful qualitative information to inform potential future fish testing requirements.
podatke o biološkim zapažanjima makromorfologije, uključujući sekundarna spolna obilježja, proizvodnju jajašaca i VTG,
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).
SMJERNICE ZA TUMAČENJE I PRIHVAĆANJE REZULTATA ISPITIVANJA
Daily quantitative observations of spawning should be recorded on a replicate basis.
Znakove bolesti i toksičnih učinaka potrebno je detaljno ocijeniti i o njima izvijestiti.
Egg production should be recorded as the number of eggs/surviving female/day on a replicate basis.
Primjerice, moguće je da na nastajanje VTG-a u ženki utječu i opća toksičnost te neendokrino toksični načini djelovanja, primjerice hepatotoksičnost.
Eggs will be removed daily from the test chambers.
Međutim, u tumačenju učinaka postiže se veća sigurnost ako postoje druge skupine, tretirane različitim koncentracijama, koje nisu narušene sustavnom toksičnošću.
Spawning substrates should be placed in the test chamber for the fathead minnow and zebrafish to enable fish to spawn in normal conditions.
Za prihvaćanje rezultata ispitivanja potrebno je razmotriti nekoliko aspekata.
It is not considered necessary to provide spawning substrate for medaka.
Primjeri raspona vrijednosti u kontrolnim i tretiranim skupinama nalaze se u izvješćima o validaciji (1., 2., 3. i 4.).
Humane killing of fish
Visoke vrijednosti VTG-a u mužjaka iz kontrolnih skupina mogu utjecati na osjetljivost testa i njegovu sposobnost otkrivanja slabih agonista estrogena.
Some endocrine active chemicals may induce changes in specialised secondary sex characteristics (number of nuptial tubercles in male fathead minnow, papillary processes in male medaka).
Što se tiče kvantifikacije proizvodnje jajašaca, ona je podložna bitnim varijacijama [koeficijent varijacije (CV) može iznositi od 20 do 60 %] koje mogu utjecati na sposobnost testa da otkrije značajno opadanje proizvodnje jajašaca manje od 70 % dok se CV približava vrijednosti od 50 % ili više.
Such gross morphological observations may provide useful qualitative and quantitative information to inform potential future fish testing requirements.
Ako laboratorij nije ranije proveo test ili ako je došlo do znatnih promjena (primjerice promjene ribljeg soja ili dobavljača), savjetuje se provedba studije tehničke osposobljenosti.
The number and size of nuptial tubercles in fathead minnow and papillary processes in medaka can be quantified directly or more practically in preserved specimens.
Preporučuje se upotreba kemikalija koje uključuju niz načina djelovanja ili učinaka na više krajnjih točaka ispitivanja.
Recommended procedures for the evaluation of secondary sex characteristics in fathead minnow and medaka are available from Appendix 5A and Appendix 5B, respectively.
U praksi se svaki laboratorij potiče na izradu vlastitih prijašnjih kontrolnih podataka za mužjake i ženke te na primjenu pozitivne kontrolne kemikalije za estrogensko djelovanje (primjerice 17β-estradiola pri 100 ng/l ili poznatog slabog agonista), čija je posljedica povećanje VTG-a u mužjaka riba, pozitivne kontrolne kemikalije za inhibiciju aromataze (primjerice fadrozol ili prokloraz pri 300 μg/l), čija je posljedica smanjenje VTG-a u ženki riba, te pozitivne kontrolne kemikalije za androgensko djelovanje (primjerice 17β-trenbolon pri 5 μg/l), čija je posljedica indukcija sekundarnih spolnih obilježja u ženki debeloglave gavčice i medake.
Vitellogenin (VTG)
Svi ti podaci mogu se usporediti s dostupnim podacima iz validacijskih studija (1., 2. i 3.) kako bi se osigurala osposobljenost laboratorija.
Blood is collected from the caudal artery/vein with a heparinised microhematocrit capillary tubule, or alternatively by cardiac puncture with a syringe.
Općenito, vrijednosti VTG-a smatraju se pozitivnima ako je došlo do statistički značajnog povećanja VTG-a u mužjaka (p < 0,05) ili statistički značajnog smanjenja u ženki (p < 0,05) najmanje pri najvećoj ispitivanoj dozi u usporedbi s kontrolnom skupinom te u izostanku znakova opće toksičnosti.
Plasma is separated from the blood via centrifugation, and stored with protease inhibitors at – 80 °C, until analysed for VTG.
Kako je prethodno navedeno, smanjenje VTG-a ne mora u potpunosti biti endokrinog podrijetla, ali pozitivan rezultat u pravilu bi trebalo tumačiti kao dokaz endokrinog djelovanja in vivo te je potrebno pokrenuti aktivnosti u svrhu dodatnih pojašnjenja.
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.
OECD (2006b).
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).
US EPA (2007).
The correlation coefficient (R2) for calibration curves should be greater than 0,99.
(2004).
However, a high correlation is not sufficient to guarantee adequate prediction of concentration in all ranges.
Andersen L., et al.
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.
(2006).
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.
Ankley G.T., et al.
If the curve is split, both line segments should have R2 > 0,99.
(2002).
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.
Panter G.H., et al.
On each day that VTG assays are performed, a fortification sample made using an inter-assay reference standard will be analysed (Appendix 7).
(2004).
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.
Parks L.G., et al.
Evaluation of gonadal histopathology
(1999).
Performance of gonadal histopathology may be required by regulatory authorities to study the target organ on the HPG axis following chemical exposure.
Fenske M., et al.
In this respect, gonads are fixed either whole body or dissected.
(2001).
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.
Holbech H., et al.
These diagnostic responses essentially include the presence of testicular oocytes, Leydig cell hyperplasia, decreased yolk formation, increased spermatogonia and perifollicular hyperplasia.
(2001).
Other gonadal lesions like oocyte atresia, testicular degeneration, and stage changes, may have various causes.
Vitellogenin induction by 17b-estradiol and 17a-ethinylestradiol in male zebrafish (Danio rerio).
The Guidance document on fish gonadal histopathology specifies procedures that will be used in the dissection, fixation, sectioning and histopathological evaluation of the gonads (22).
C. 131: 531-539.
Where a solvent control is used, an appropriate statistical test should be performed between the dilution water and solvent controls for each endpoint.
Development and validation of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio rerio).
Guidance on how to handle dilution water and solvent control data in the subsequent statistical analysis can be found in OECD, 2006c (28).
Yokota H., et al.
All biological response data should be analysed and reported separately by sex.
(2001).
If the required assumptions for parametric methods are not met — non-normal distribution (e.g. Shapiro-Wilk's test) or heterogeneous variance (Bartlett's test or Levene's test), consideration should be given to transforming the data to homogenise variances prior to performing the ANOVA, or to carrying out a weighted ANOVA.
Development of an ELISA for determination of the hepatic vitellogenin in Medaka (Oryzias latipes).
Dunnett's test (parametric) on multiple pair-wise comparisons or a Mann-Whitney with Bonferroni adjustment (non-parametric) may be used for non-monotonous dose-response.
Effects of the androgenic growth promoter 17-beta-trenbolone on fecundity and reproductive endocrinology of the fathead minnow.
Other statistical tests may be used (e.g. Jonckheere-Terpstra test or Williams test) if the dose-response is approximately monotone.
OECD (2010).
A statistical flowchart is provided in Appendix 8 to help in the decision on the most appropriate statistical test to be used.
Guidance Document on Fish Gonadal Histopathology.
Reporting of test results
123, Pariz.
Study data should include:
OECD (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.
Testing facility:
Series on Testing and Assessment No.
Responsible personnel and their study responsibilities
Pariz
Each laboratory should have demonstrated proficiency using a range of representative chemicals
Aquatic Toxicology, 76; pp. 69–92.