natgillin/translations-raw huggingface dataset:
If samples are stored to be analysed at a later time, the storage method of the samples should be previously validated.
vzorci za analizo VTG se vstavijo v mikroepruvete in takoj zamrznejo v tekočem dušiku;
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.
trup, vključno s spolnimi žlezami, se vstavi v predhodno označene plastične kasete za tkivo, ki se prenesejo v Davidsonov ali Bouinov fiksativ.
During the test, dissolved oxygen, pH, total hardness, conductivity, salinity (if relevant), and temperature should be measured in all test chambers.
Fiksativa mora biti vsaj desetkrat več od približne prostornine tkiv.
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.
Posoda s fiksativom se pet sekund nežno pretresa, da se zračni mehurčki odstranijo iz kasete;
Temperature should preferably be monitored continuously in at least one test chamber.
vsa tkiva ostanejo v Davidsonovem fiksativu čez noč, naslednji dan pa se prenesejo v posamezne posode z 10-odstotnim nevtralno pufranim formalinom.
Results should be based on measured concentrations.
Posode s kasetami se 5 sekund nežno stresajo, da se zagotovi ustrezna penetracija formalina v kasete;
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.
tkiva ostanejo v Bouinovem fiksativu 24 ur, sledi pa prenos v 70-odstotni etanol.
Observations and measurements
Obdelava tkiva
Stage of embryonic development
dehidracija tkiva za ustrezno penetracijo parafina;
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.
impregnacija tkiva s parafinom, da se ohrani neokrnjenost tkiva in ustvari trdna podlaga za mikrotomijo;
Hatching and survival
izbere se časovni načrt obdelave;
for eggs:
košare s kasetami se odstranijo iz naprave za obdelavo in potopijo v s parafinom napolnjeno prednjo komoro grelne naprave za vstavljanje ali pa se kasete prestavijo v ločen parafinski grelnik;
for larvae and juvenile fish:
Pokrov kasete se odstrani in zavrže, na oznaki kasete pa se preverijo zabeležke o živali, da se pred vstavljanjem odpravijo morebitne neskladnosti;
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.
izbere se ustrezno velik kalup za vstavljanje;
Abnormal appearance
kalup se drži pod cevko dozirne konzole in napolni s tekočim parafinom;
The number of larvae or fish showing abnormality of body form should be recorded, and the appearance and the nature of the abnormality described.
vzorec se odstrani iz kasete in vstavi v stopljeni parafin v kalupu.
It should be noted that abnormal embryos and larvae occur naturally and can be of the order of several per cent in the control(s) in some species.
To se ponovi pri 4–8 vzorcih za vsak kalup s parafinom.
Abnormal animals should only be removed from the test chambers on death.
Mesto posamezne ribe se označi tako, da se prva riba položi pod kotom 180 stopinj glede na ribo 2–4/8;
However, in accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes, if abnormalities result in pain, suffering and distress or lasting harm, and death can be reliably predicted, animals should be anaesthetised and euthanised according to the description in paragraph 44 and treated as mortality for data analysis..
vzorec se prekrije z dodajanjem dodatnega parafina;
Abnormal behaviour
kalup s podlago kasete se postavi na hladilno ploščo kriogene posode;
At the end of the test all surviving fish should be euthanised (anaesthetised if blood samples should be taken), and individual wet weight (blotted dry) should be measured.
rezanje in pritrditev histoloških rezin za barvanje.
These observations will result in some or all of the following data being available for reporting:
vpenjalo se z vrtenjem kolesa mikrotoma pomika naprej, s parafinske površine bloka pa se režejo debele rezine, dokler nož ne doseže vstavljenih tkiv;
cumulative mortality;
debelina rezine se na mikrotomu nastavi na 3–5 mikronov.
numbers of healthy fish at end of test;
Vpenjalo se pomika naprej in z bloka se odreže več rezin, da se odstranijo vsi artefakti, ki so med grobimi rezi nastali na rezalni površini tkiva;
time to start of hatching and end of hatching;
blok se lahko odstrani iz vpenjala in s srednjo stranjo naprej položi na led, da se tkivo zmoči;
length and weight of surviving animals;
naslednja faza mikrotomije je končno rezanje rezin in pritrditev tkivnih rezin na mikroskopske preparate.
numbers of deformed larvae;
Ti postopki se izvedejo, kot sledi:
numbers of fish exhibiting abnormal behaviour.
če je bil blok postavljen na led, se odstrani z njega in vstavi v vpenjalo mikrotoma;
Sampling of fish
rezine se plosko položijo na vodno gladino v vodni kopeli.
Fish sampling is performed at termination of the test.
Poskusi se obdržati vsaj eno rezino brez gub in zračnih mehurčkov, ujetih pod njo;
Sampled fish should be euthanised with e.g. MS-222 (100-500 mg per l buffered with 200 mg NaHCO3 per l) or FA-100 (4-allyl-2-methoxyphenol: eugenol) and individually measured and weighed as wet weight (blotted dry) or anaesthetised if a blood sample should be taken (see paragraph 49).
mikroskopsko objektno stekelce se potopi pod najboljšo rezino, ki se s stekelcem dvigne iz vode.
Sampling for VTG analysis and sex determination via histological evaluation
Ta postopek se imenuje ‚pritrditev‘ rezine na preparat;
All fish should be sampled and prepared for analysis of sex and VTG.
za sklop rib se pripravijo tri rezine.
All fish should be analysed histologically to determine sex.
Druga in tretja rezina se odrežeta v razmiku 50 mikronov za prvo rezino.
For the VTG measurements, a sub-sampling of at least 16 fish from each replicate is accepted.
Če ribe niso vstavljene s spolnimi žlezami v isti rezalni ravnini, je treba narediti več rezin, da se zagotovi vsaj šest rezin s spolnimi žlezami za vsako ribo;
More fish should be analysed for VTG if the results of the sub-sampling turn out to be unclear.
s pisalom za označevanje preparatov se na preparatu označi številka bloka, iz katerega je bil preparat ustvarjen;
The sampling procedure for VTG and sex determination is dependent on the VTG analysis method:
preparat se položi na podstavek za barvanje;
Head/tail homogenate method for VTG analysis
blok se odstrani iz vpenjala in shrani s sprednjim delom naprej.
The fish is euthanised.
Barvanje, namestitev krovnega stekelca in označevanje preparatov
Head and tail of each fish are separated from the body of the fish by cuts made right behind the pectoral fins, and right behind the dorsal fin, using a scalpel (See Figure 1).
obarvanje rezin za histopatološko preiskavo;
The head and tail part from each fish are pooled, weighed and individually numbered, frozen in liquid nitrogen and stored at – 70° or less for VTG analysis.
trajno zatesnjeno pritrjena in obarvana tkiva;
The body part of the fish is numbered and fixed in an appropriate fixative for histological evaluation (22).
trajno identificirane obarvane rezine, ki omogočajo popolno sledljivost;
By use of this method VTG and histopathology are evaluated on each individual and a possible change in the VTG level can thus be related to the phenotypic sex of the fish or genetic sex (Japanese medaka and the three-spined stickleback) of the fish.
barvanje:
For further information see guidance for homogenisation (Appendix 5) and guidance for VTG quantification (Appendix 6).
preparati se pred barvanjem čez noč sušijo na zraku;
Liver homogenate method for VTG analysis
rezine se barvajo s hematoksilin-eozinom;
The liver is dissected out and stored at – 70 °C or below.
namestitev krovnega stekelca:
Recommended procedures for liver excision and pre-treatment are available in OECD TG 229 (37) or Chapter C.37 of this Annex (38).
krovna stekelca se lahko namestijo ročno ali samodejno;
Livers are then individually homogenised as described in OECD TG 229 or Chapter C.37 of this Annex.
preparat se pomoči v ksilen ali TissueClear, odvečni ksilen/TissueClear pa se nežno odstrani s preparata;
The supernatant collected is used for measuring VTG with a homologous ELISA technique (see Appendix 6 for an example of quantification in zebrafish or OECD TG 229 (37) for Japanese medaka).
v bližino konca preparata nasproti zamrznjenega konca ali na krovno stekelce se nanese približno 0,1 ml pritrditvenega medija;
Following this approach, it is also possible to have individual fish data on both VTG and gonad histology.
krovno stekelce se med nameščanjem na preparat nagne pod majhnim kotom;
Blood plasma method for VTG analysis
označevanje:
Blood is collected from the anaesthetised fish by cardiac puncture, caudal vein or tail cutting, and centrifuged at 4 °C for plasma collection.
vsaka oznaka preparata mora vsebovati naslednje informacije:
The plasma is stored at – 70 °C or below until use.
ime laboratorija;
The whole fish is euthanised and fixed for histology.
št. vzorca/št.
Both plasma samples and fish are numbered individually to relate VTG levels to the sex of the fish.
Preparata;
How to cut a fish for measurement of VTG in head/tail homogenate and histological evaluation of the mid section
kemikalijo/skupino tretiranja;
Genetic sex determination
Statistični diagram poteka za analizo vitelogenina Ne Da Ne Da Ne Da Ne
A biological sample for the determination of the genetic sex is taken from individual fish in species possessing appropriate markers.
Tamhane-Dunnettov preskus
For Japanese medaka, the anal fin or dorsal fin is collected.
Tamhane-Dunnettov preskus ugnezdene analize varianc
A detailed description is given in Appendix 9 including tissue sampling and sex determination by a PCR-method.
Sredine ponovitev niso normalne ali niso homogene.
Equally, for the three spined stickleback, a description of tissue sampling and a sex determining PCR-method is given in Appendix 10.
Sredine ponovitev niso normalno porazdeljene.
VTG measurement
Ugnezdena analiza varianc ni normalna.
The measurement of VTG should be based upon a quantitative and analytically validated method.
Dunnettov preskus
Information should be available upon the intra-assay and inter-assay variability of the method used in a given laboratory.
Transformacija za stabilizacijo varianc?
Considering the variability of VTG measurement, NOECs based on this endpoint alone should be treated with great care.
Monoton
Different methods are available to assess VTG production in the fish species considered in this assay.
>=4 pon. na konc. <=3 pon. na konc.
A measurement technique that is both relatively sensitive and specific is the determination of protein concentrations via enzyme-linked immunosorbent assay (ELISA).
Dunnov preskus >=4 pon. na konc.
Homologous antibodies (raised against VTG of the same species) and most important homologous standards should be used.
Regresivni Jonckheerov ali Williamsov preskus
Sex determination
Združite kontrole, ohranite podskupine
Dependent on the VTG sampling procedure, whole fish or the remaining mid-section of each fish is placed in a pre-labelled processing cassette and fixed in an appropriate fixative for histological determination of sex (optionally also for evaluation of gonadal staging).
Primerjajte kontrole z Wilcoxonovim ali t-preskusom.
Guidance on fixation and embedding is provided in Appendix 7 as well as in the OECD Guidance Document on the Diagnosis of Endocrine-Related Histopathology of Fish Gonads (22).
Ali se kontrole razlikujejo?
The individuals should be placed longitudinally in the paraffin block.
Da
At least six longitudinal sections (3-5 μm in thickness) in a frontal plane including gonadal tissue from both gonads are taken from each individual.
Ali so podatki normalno porazdeljeni?
The interval between these sections should be approximately 50 μm for males and 250 μm for females.
Ali so podatki skladni z monotonim odzivom na odmerek?
However, since each block will often contain males and females (if more than one individual are embedded in each block), the interval between sections from these blocks should be approximately 50 μm until at least six sections of the gonads from each male are obtained.
(‡) Ali izbira druge dogovorjene kontrole.
Thereafter, the interval between sections can be increased to approximately 250 μm for the females.
Po arkus sinus-korenski transformaciji.
Sections are stained with haematoxylin and eosin and examined by light-microscopy with focus on sex (male, female, intersex or undifferentiated).
(+) Pri manj kot 5 poskusnih enotah na tretiranje je treba uporabiti preskus po J-T ali M-W preskus, če je na voljo.
Intersex is defined as presence of more than one oocyte in testis per six sections analysed or spermatogenic cells (yes/no) in ovaries.
Kontrole se združijo.
Histopathology and staging of ovaries and testis is optional but if investigated, the results should be statistically analyzed and reported.
Izpusti se kontrola z vodo (‡).
It should be noted that some fish species naturally lack a fully developed pair of gonads and only one gonad may be present (e.g. Japanese medaka and occasionally zebrafish).
Kontrole se primerjajo s t-preskusom.
All such observations should be recorded.
Dodatek 9
Genetic sex determination in individual Japanese medaka is based on the presence or absence of the medaka male-sex determining gene, DMY, which is located on the Y chromosome.
Smernice za vzorčenje tkiva za določitev genetskega spola in določitev genetskega spola z metodo PCR
The genotypic sex of medaka can be identified by sequencing the DMY gene from DNA extracted from for instance a piece of anal fin or dorsal fin.
Vzorčenje tkiva, priprava in shranjevanje pred določanjem genetskega spola z metodo PCR pri medaki (pripravil laboratorij za vodne organizme družbe Bayer CropScience AG)
The presence of DMY indicates a XY (male) individual regardless of phenotype, while the absence of DMY indicates a XX (female) individual regardless of phenotype (23).
Pri vsaki ribi se z natančnimi škarjami odrežeta predrepna in hrbtna plavut in vstavita v epruveto s 100 μl ekstrakcijskega pufra 1 (podrobnosti o pripravi pufra so navedene v nadaljevanju).
Guidance for tissue preparation and PCR method is given in Appendix 9.
Po rezanju vsake ribe se škarje očistijo v čaši z destilirano H2O in osušijo s papirnato brisačo.
The genetic sex determination in individual three-spined stickleback is also performed via a PCR method, described in Appendix 10.
Tkiva plavuti se za lizo celic homogenizirajo s teflonskim pestičem mikroepruvete.
The occurrence of intersex (for definition, see Appendix 1) should be reported.
Za vsako epruveto se uporabi novo pestilo, da se prepreči onesnaženje.
Secondary sexual characteristics
Pestila se čez noč pustijo v 0,5 M NaOH, 5 minut spirajo v destilirani H2O ter do uporabe shranijo v etanolu ali sterilizirajo po avtoklaviranju.
Secondary sexual characteristics are under endocrine control in species like the Japanese medaka;
Tkivo plavuti se lahko shrani tudi brez ekstrakcijskega pufra 1 na suhem ledu in nato v zamrzovalniku pri – 80 °C, da se prepreči degeneracija DNA.
therefore observations of physical appearance of the fish should if possible be made at the end of the exposure.
Vendar ekstrakcija poteka bolje, če se DNA ekstrahira hkrati (za ravnanje glej zgoraj; vzorce je treba odtajati na ledu po shranjevanju pri – 80 °C, preden se v epruvete doda pufer).
In the Japanese medaka, the papillary formation on the posterior part of the anal fin in females is androgen sensitive.
Po homogenizaciji se vse epruvete postavijo v vodno kopel, kjer 15 minut vrejo pri 100 °C.
Chapter C.37 of this Annex (38) provides relevant photographs of male secondary sex characteristics and androgenised females.
Nato se s pipeto v vsako epruveto doda 100 μl ekstrakcijskega pufra 2 (podrobnosti o pripravi pufra so navedene v nadaljevanju).
It is important that the strongest valid statistical test determine the endpoint.
Vzorci se pri sobni temperaturi hranijo 15 minut in se pri tem občasno nežno ročno pretresejo.
The replicate is the experimental unit but intra-replicate variability should be included in the statistical testing.
Po tem se vse epruvete ponovno postavijo v vodno kopel, kjer še 15 minut vrejo pri 100 °C.
A decision flow-chart is available in Appendix 8 to help with the most appropriate statistical test to use based on the characteristic of the data obtained from the test.
Epruvete se do nadaljnje analize zamrznejo pri – 20 °C.
Statistical significance level is 0,05 for all endpoints included.
Priprava pufra
Proportions of sex and genetic sex
Pufer PCR 1:
The proportions of sex should be analysed for significant effect (NOEC/LOEC approach) of exposure by Jonckheere-Terpstra (Trend test) if a monotone dose-response exists.
500 mg N-lavroilsarkozina (npr. Merck KGaA, Darmstadt, GE)
If non-monotonicity is found then a pair wise test should be applied:
→ avtoklav
Use Dunnett's test if normality and homogenous variance can be obtained.
Pufer PCR 2:
Use Tamhane-Dunnett if heterogeneous variance is present.
20 g Chelex (npr. Biorad, Munich, GE)
Otherwise use exact Mann-Whitney test with Bonferroni-Holm adjustment.
Določanje genetskega spola (z metodo PCR) pri medaki (pripravila laboratorij za vodne organizme družbe Bayer CropScience AG in Universität Würzburg Biozentrum)
A flow chart describing the statistics of the proportions of sex is placed in Appendix 8.
Pripravljene in zamrznjene epruvete (opisano v zgornjem oddelku) se odtajajo na ledu.
The proportions of sex should be presented in tables as concentration proportions ± SD of males, females, intersex and undifferentiated.
Nato se centrifugirajo v mikrocentrifugirki (30 sekund pri največji hitrosti in sobni temperaturi).
Statistical significance should be highlighted.
Za PCR se uporabi čisti supernatant iz oborine.
Examples are presented in the FSDT Phase 2 validation report (42).
Preprečiti je treba vsak prenos sledi Chelexa (lokaliziranega v oborini) v reakcijo PCR, saj bi to vplivalo na aktivnost Taq-polimeraze.
Genetic sex should be reported as percentage of phenotypic sex reversal of males, females, intersex and undifferentiated.
Supernatant se uporabi neposredno ali pa se zamrzne (pri – 20 °C) in odtaja v več ciklih brez negativnega učinka na DNA za poznejše analize.
VTG concentrations
Priprava ‚reakcijske zmesi‘ (25 μl na vzorec):
VTG concentrations should be analysed for significant effect (NOEC/LOEC approach) of exposure.
Prostornina
The Dunnett test is preferable to the t-test with Bonferroni correction.
Končna koncentracija
Where a Bonferroni correction is used, the Bonferroni-Holm correction is preferable.
Matrična DNA
Allowance should be made for log-transformation of VTG to achieve normality and variance homogeneity.
0,5 μl–2 μl
Next, if the concentration-response is consistent with monotonicity, then the JonckheereTerpstra test is preferable to any of the above.
10 × pufer PCR z MgCl2
If t-tests or Dunnett's test is used, there is no need for a ANOVA significance F-test in order to proceed.
2,5 μl
For details see the flow chart in Appendix 8.
Nukleotidi (vsi dATP, dCTP, dGTP, dTTP)
Results should be reported in tables as concentration means ± SD for males, females, intersex and undifferentiated separately.
Smerni začetni oligonukleotid (10 μM) (glej v nadaljevanju 3–5)
Statistical significance for phenotypic females and phenotypic males should be highlighted.
0,5 μl
Test chemical actual concentrations
Protismerni začetni oligonukleotid (10 μM) (glej v nadaljevanju 3–5)
The actual chamber concentrations of the test chemical should be analysed in frequencies described in paragraph 34.
Voda (stopnje PCR)
Results should be reported in tables as mean concentration ± SD on replicate basis as well as on concentration basis with information on number of samples and with outliers from the mean treatment concentration ± 20 % highlighted.
do 25 μl
Examples can be found in the FSDT Phase 2 validation report (42).
Taq E-polimeraza
The test results should be interpreted with caution where measured test chemical concentrations in test solutions occur at levels near the detection limit of the analytical method.
0,3 μl
Relevant physical-chemical properties;
10 × pufer PCR z MgCl2:
chemical identification data including purity and analytical method for quantification of the test chemical.
670 mM Tris/HCl (pH 8,8 pri 25 °C), 160 mM (NH4)2SO4, 25 mM MgCl2, 0,1 % Tween 20
Test procedure used (e.g. flow-through semi-static/renewal);
Za vsako PCR (glej v nadaljevanju 3–5) je potreben poseben začetni oligonukleotid kot nova kombinacija ‚reakcijske zmesi‘ in ustrezne količine matrične DNA, ki je potrebna za vsak vzorec (glej zgoraj).
test design including test concentrations, method of preparation of stock solutions (in an Annex), frequency of renewal (the solubilising agent and its concentration should be given, when used);
Ustrezne prostornine se s pipetami prenesejo v nove epruvete.
The nominal test concentrations, the means of the measured values and their standard deviations in the test chambers and the method by which these were attained (the analytical method used should be presented in an Annex);Evidence that the measurements refer to the concentrations of the test chemical in true solution;
Po tem se vse epruvete zaprejo, mešajo (približno 10 sekund) in centrifugirajo (10 sekund pri sobni temperaturi).
Water quality within test chambers:
Nato se lahko začnejo ustrezni programi PCR.
Detailed information on feeding (e.g. type of food(s), source, amount given and frequency and analyses for contaminants (e.g. PCBs, PAHs and organochlorine pesticides) if relevant.
V vsakem programu PCR se uporabita še pozitivna kontrola (reprezentativen vzorec DNA z znano aktivnostjo in jasnimi rezultati) in negativna kontrola (1 μl destiliranega H2O).
Evidence that controls met the validity criteria:
Priprava agaroznega gela (1 %) – med potekom programov PCR
data on hatching rate should be presented in tables as percentage per replicate and per concentration.
3 g agaroze se raztopijo v 300 ml pufra 1 × TAE (1-odstotni agarozni gel);
Outliers from the acceptance criteria (in controls) should be highlighted.
raztopina mora vreti v mikrovalovni pečici (približno 2–3 minute);
Survival should be presented as percentage per replicate and per concentration.
vročo raztopino je treba preliti v model za vlivanje, ki stoji na ledu;
Outliers from the validity criteria (in controls) should be highlighted;
po približno 20 minutah je agarozni gel pripravljen za uporabo;
Clear indication of the results obtained on the different endpoints observed:
agarozni gel se do konca programov PCR shrani v pufru 1 × TAE.
embryo survival and hatching success;
Program PCR za aktin:
external abnormalities;
namen te reakcije PCR je dokazati, da se DNA v vzorcu ne poškoduje.
length and weight;
Poseben začetni oligonukleotid:
VTG measurements (ng/g homogenate, ng/ml plasma or ng/mg liver);
‚Mact1(zgornji/smerni)‘ → TTC AAC AGC CCT GCC ATG TA
gonadal histology, sex ratio, genetic sex data;
‚Mact2(spodnji/protismerni)‘ → GCA GCT CAT AGC TCT TCT CCA GGG AG
incidence of any unusual reactions by the fish and any visible effects produced by the test chemical.
Program:
The results should be presented as mean values ± standard deviation (SD) or standard error (SE).
Cikel (35-krat):
Statistics should be reported as a minimum as NOEC and LOEC and confidence intervals.
Denaturacija
The statistical flow chart (Appendix 8) should be followed.
→ 45 sekund pri 95 °C
Sumpter, 1996, “Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals”, Environmental Toxicology and Chemistry 15, pp. 194-202.
Prileganje