natgillin/translations-raw huggingface dataset:
Abnormal animals should only be removed from the test chambers on death.
Homogeenimispuhver:50 mM Tris-HCl pH 7,4;
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..
1 % proteaasiinhibiitorite segu (Sigma):
Abnormal behaviour
12 ml Tris-HCl pH 7,4 + 120 μl proteaasiinhibiitorite segu (või samaväärset proteaasiinhibiitorite segu).TRIS:
Abnormalities, e.g. hyperventilation, uncoordinated swimming, atypical quiescence and atypical feeding behaviour should be recorded at appearance.
TRIS-ULTRA PURE (ICN)Proteaasiinhibiitorite segu:
Weight
Sigmalt (imetajate kudede jaoks), tootenumber P 8340.
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.
Juhend vitellogeniini mõõtmiseks vöötdaanio (Danio rerio) pea/saba homogenaadist (muudetud holbech et al., 2001). võib kasutada muid meetodeid, milles kasutatakse homoloogseid antikehi ja standardeid
Length
Mikrotiiterplaadid (sertifitseeritud Maxisorp F96, Nunc, Roskilde, Taani), mis on eelnevalt kaetud vöötdaanio lipovitelliini vastase IgG-ga kontsentratsioonis 5 μg/ml, sulatatakse ja pestakse kolm korda pesemispuhvriga (*).
At the end of the test, individual lengths (standard length) should be measured.
Vöötdaanio puhastatud vitellogeniini standardit [1] lahjendatakse seeriana kontsentratsioonideni 0,2, 0,5, 1, 2, 5, 10, 20 ng 1 ml lahjenduspuhvri (**) kohta ja proove lahjendatakse vähemalt 200 korda (maatriksiefekti vältimiseks) lahjenduspuhvris ja kantakse plaatidele.
These observations will result in some or all of the following data being available for reporting:
Kontrollkatse tehakse kahe paralleelkatsena.
cumulative mortality;
Mikrotiiterplaadi igasse süvendisse kantakse 150 μl.
numbers of healthy fish at end of test;
Standardainega tehakse kaks paralleelmääramist ja proovidega kolm.
time to start of hatching and end of hatching;
Inkubeeritakse 4 °C juures üle öö loksutil.
length and weight of surviving animals;
Plaate pestakse 5 korda pesemispuhvriga (*).
numbers of deformed larvae;
Dekstraaniahelale (nt AMDEX A/S, Taani) seotud mädarõika peroksidaas ja konjugeeritud antikehad lahjendatakse pesemispuhvris;
numbers of fish exhibiting abnormal behaviour.
kasutatav lahjendus sõltub partiist ja vanusest.
Sampling of fish
Igasse süvendisse pannakse 150 μl ja plaate inkubeeritakse loksutil 1 tund toatemperatuuril.
Fish sampling is performed at termination of the test.
Plaate pestakse viis korda pesemispuhvriga (*) ja süvendite põhjad puhastatakse hoolikalt etanooliga.
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).
Mikrotiiterplaadi igasse süvendisse kantakse 150 μl lahust TMB plus (***).
Sampling for VTG analysis and sex determination via histological evaluation
Plaat varjatakse valguse eest fooliumiga ja jälgitakse värvuse ilmumist loksutil.
All fish should be sampled and prepared for analysis of sex and VTG.
Kui standardkõver on täielikult ilmutatud, pidurdatakse ensüümi aktiivsus 150 μl 0,2 M H2SO4 lahuse lisamisega igasse süvendisse.
All fish should be analysed histologically to determine sex.
Pesemispuhver:
For the VTG measurements, a sub-sampling of at least 16 fish from each replicate is accepted.
PBS-põhilahust (****)
More fish should be analysed for VTG if the results of the sub-sampling turn out to be unclear.
pH viiakse 7,3 peale ja täidetakse 5 l-ni veega, mis on lastud läbi Millipore'i filtri.
The sampling procedure for VTG and sex determination is dependent on the VTG analysis method:
Säilitatakse temperatuuril 4 °C.
Head/tail homogenate method for VTG analysis
Lahjenduspuhver:
The fish is euthanised.
PBS-põhilahus (****)
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).
pH viiakse 7,3 peale ja täidetakse 1 l-ni veega, mis on lastud läbi Millipore'i filtri.
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.
TMB plus on kasutusvalmis substraat, mida toodab KemEnTec (Taani).
The body part of the fish is numbered and fixed in an appropriate fixative for histological evaluation (22).
See on tundlik valguse suhtes.
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.
PBS-põhilahust
For further information see guidance for homogenisation (Appendix 5) and guidance for VTG quantification (Appendix 6).
pH viiakse 6,8 peale ja täidetakse 2 l-ni veega, mis on lastud läbi Millipore'i filtri.
Liver homogenate method for VTG analysis
Säilitatakse toatemperatuuril.
The liver is dissected out and stored at – 70 °C or below.
Juhend koelõikude ettevalmistamiseks soo ja sugunäärmete arengustaadiumi määramiseks
Recommended procedures for liver excision and pre-treatment are available in OECD TG 229 (37) or Chapter C.37 of this Annex (38).
Käesoleva osa eesmärk on kirjeldada koelõikude ettevalmistamist histoloogiliseks hindamiseks.
Livers are then individually homogenised as described in OECD TG 229 or Chapter C.37 of this Annex.
Võib kasutada ka muid meetodeid, millega saadakse sarnased soolise jaotumise ja sugunäärmete arengustaadiumi näitajad.
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).
Väheste eranditega on need meetodid samasugused jaapani riisikala ja vöötdaanio puhul.
Following this approach, it is also possible to have individual fish data on both VTG and gonad histology.
Valutu surmamine, lahkamine ja kudede fikseerimine
Blood plasma method for VTG analysis
Eesmärgid:
Blood is collected from the anaesthetised fish by cardiac puncture, caudal vein or tail cutting, and centrifuged at 4 °C for plasma collection.
Tagada kalade humaanne surmamine.
The plasma is stored at – 70 °C or below until use.
Teha vajalikud kehamassi ja muud mõõtmised.
The whole fish is euthanised and fixed for histology.
Hinnata sekundaarseid sootunnuseid.
Both plasma samples and fish are numbered individually to relate VTG levels to the sex of the fish.
Lõigata välja koed vitellogeniini määramiseks.
How to cut a fish for measurement of VTG in head/tail homogenate and histological evaluation of the mid section
Fikseerida sugunäärmed.
Genetic sex determination
Töö käik:
A biological sample for the determination of the genetic sex is taken from individual fish in species possessing appropriate markers.
Kalad tuleks surmata vahetult enne lahkamist.
For Japanese medaka, the anal fin or dorsal fin is collected.
Kui lahkajaid ei ole palju, siis ei tohiks korraga surmata paljusid kalu.
A detailed description is given in Appendix 9 including tissue sampling and sex determination by a PCR-method.
Väikse kahvaga võetakse kala katsekambrist ja viiakse transpordimahutis lahkamiskohta.
Equally, for the three spined stickleback, a description of tissue sampling and a sex determining PCR-method is given in Appendix 10.
Kala pannakse valutu surmamise lahusesse.
VTG measurement
Kala võetakse lahusest välja, kui hingamisliigutused on lakanud ja kala ei reageeri välistele ärritajatele.
The measurement of VTG should be based upon a quantitative and analytically validated method.
Kala kaalutakse märjalt.
Information should be available upon the intra-assay and inter-assay variability of the method used in a given laboratory.
Selleks, et valmistada koed ette vitellogeniini määramiseks, võib kala panna lahkamismikroskoobi korkkattega alusele.
Considering the variability of VTG measurement, NOECs based on this endpoint alone should be treated with great care.
Vöötdaanio puhul lõigatakse pea maha otse rinnauime tagant ja saba lõigatakse maha otse seljauime tagant.
Different methods are available to assess VTG production in the fish species considered in this assay.
b.
A measurement technique that is both relatively sensitive and specific is the determination of protein concentrations via enzyme-linked immunosorbent assay (ELISA).
Jaapani riisikala puhul avatakse kõht hoolika sisselõikega, mis ulatub mööda kõhu keskjoont rinnavöötmest kuni punktini, mis asub kohe päraku peapoolse otsa kõrval.
Homologous antibodies (raised against VTG of the same species) and most important homologous standards should be used.
Väikeste pintsettide ja väikeste kääridega eemaldatakse hoolikalt maks.
Sex determination
Proov vitellogeniini määramiseks pannakse Eppendorfi katsutisse ja külmutatakse viivitamata vedelas lämmastikus.
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).
Rümp sugunäärmetega pannakse eelnevalt märgistatud plastikust koekassetti, mis paigutatakse Davidsoni või Bouini fikseerivasse lahusesse.
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).
Fikseerivat lahust peaks ruumala poolest olema vähemalt 10 korda rohkem kui on koe hinnanguline maht.
The individuals should be placed longitudinally in the paraffin block.
a.
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.
Kõik koed jäetakse Davidsoni fikseerivasse lahusesse üheks ööks, järgmisel päeval kantakse need üle eraldi nõudesse, milles on neutraalse puhvriga 10 % formaliini lahus.
The interval between these sections should be approximately 50 μm for males and 250 μm for females.
Nõusid kassettidega loksutatakse ettevaatlikult 5 sekundit, et formaliin saaks vabalt tungida kassettidesse.
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.
b.
Thereafter, the interval between sections can be increased to approximately 250 μm for the females.
Koed jäetakse Bouini fikseerivasse lahusesse 24 tunniks, seejärel viiakse need üle 70 % etanooli.
Sections are stained with haematoxylin and eosin and examined by light-microscopy with focus on sex (male, female, intersex or undifferentiated).
Kudede töötlemine
Intersex is defined as presence of more than one oocyte in testis per six sections analysed or spermatogenic cells (yes/no) in ovaries.
Veetustada kude parafiini korraliku läbitungimise võimaldamiseks.
Histopathology and staging of ovaries and testis is optional but if investigated, the results should be statistically analyzed and reported.
Kude immutatakse parafiiniga koe terviklikkuse säilitamiseks ja kindla pinna loomiseks, mis võimaldaks teha mikrotoomilõikeid.
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).
Märgistatud koekassetid võetakse formaliinist/etanoolist välja ja pannakse töötlemiskorvi(desse).
All such observations should be recorded.
Töötlemiskorv pannakse koeprotsessorisse.
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.
Valitakse töötlemisrežiim.
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.
Kui seade lõpetab töötlemistsükli, võib korvi(d) üle viia sisestamisseadmesse.
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).
Sisestamine
Guidance for tissue preparation and PCR method is given in Appendix 9.
Eesmärk:
The genetic sex determination in individual three-spined stickleback is also performed via a PCR method, described in Appendix 10.
Orienteerida proov tahkestatud parafiinis õigesti mikrotoomia jaoks.
The occurrence of intersex (for definition, see Appendix 1) should be reported.
Korv(id) kassettidega võetakse protsessorist välja ja sukeldatakse sisestamisseadme termokonsooli parafiiniga täidetud eeskambrisse või viiakse kassetid üle eraldi parafiinikuumutisse.
Secondary sexual characteristics
Esimene sisestatav kassett võetakse välja termokonsooli eeskambrist või parafiinikuumutist.
Secondary sexual characteristics are under endocrine control in species like the Japanese medaka;
Kasseti kaas võetakse ära ja visatakse minema ning kassetil olevat märgistust võrreldakse looma kohta registreeritud andmetega, et lahendada võimalikud vastuolud enne sisestamist.
therefore observations of physical appearance of the fish should if possible be made at the end of the exposure.
Valitakse sobiva suurusega sisestamisvorm.
In the Japanese medaka, the papillary formation on the posterior part of the anal fin in females is androgen sensitive.
Vormi hoitakse dosaatorkonsooli väljalaskeava all ja see täidetakse sula parafiiniga.
Chapter C.37 of this Annex (38) provides relevant photographs of male secondary sex characteristics and androgenised females.
Proov võetakse kassetist ja pannakse vormi sulasse parafiini.
It is important that the strongest valid statistical test determine the endpoint.
Nii tehakse iga parafiinivormi puhul 4–8 prooviga.
The replicate is the experimental unit but intra-replicate variability should be included in the statistical testing.
Iga üksiku kala asend märgitakse nii, et kala nr 1 on 180 kraadi all kala nr 2-4/8 suhtes.
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.
Lisatakse parafiini proovi katmiseks.
Statistical significance level is 0,05 for all endpoints included.
Vorm kassetialusega pannakse krüokonsooli jahutusplaadile.
Proportions of sex and genetic sex
Pärast parafiini tahkestumist võetakse plokk (st tahkestunud parafiin, mis sisaldab kudesid ja kassetipõhjasid) vormist välja.
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.
Mikrotoomia
If non-monotonicity is found then a pair wise test should be applied:
lõigata ja seada histoloogilised koelõiked valmis värvimiseks.
Use Dunnett's test if normality and homogenous variance can be obtained.
Mikrotoomia algetapil proov positsioneeritakse järgmiselt:
Use Tamhane-Dunnett if heterogeneous variance is present.
a.
Otherwise use exact Mann-Whitney test with Bonferroni-Holm adjustment.
Parafiiniplokk pannakse mikrotoomi kelgule.
A flow chart describing the statistics of the proportions of sex is placed in Appendix 8.
b.
The proportions of sex should be presented in tables as concentration proportions ± SD of males, females, intersex and undifferentiated.
Kelku liigutatakse edasi mikrotoomi ratta abil ja parafiiniploki pinnalt lõigatakse pakse kihte, kuni nuga jõuab sisestatud kudedeni.
Statistical significance should be highlighted.
c.
Examples are presented in the FSDT Phase 2 validation report (42).
Mikrotoomi lõikepaksuseks seatakse 3–5 mikronit.
Genetic sex should be reported as percentage of phenotypic sex reversal of males, females, intersex and undifferentiated.
Kelku lükatakse edasi ja plokist tehakse mitu lõiget, et kõrvaldada kõik artefaktid, mis võivad olla tekkinud koelõigu pinnal jämeda kärpimisega.
VTG concentrations
d.
VTG concentrations should be analysed for significant effect (NOEC/LOEC approach) of exposure.
Ploki võib kelgult maha võtta ja panna jääle esiküljega allapoole koe niisutamiseks.
The Dunnett test is preferable to the t-test with Bonferroni correction.
Mikrotoomia järgmine etapp on lõplik lõigete tegemine ja koeliistakute panemine objektiklaasile.
Where a Bonferroni correction is used, the Bonferroni-Holm correction is preferable.
See toimub järgmiselt:
Allowance should be made for log-transformation of VTG to achieve normality and variance homogeneity.
a.
Next, if the concentration-response is consistent with monotonicity, then the JonckheereTerpstra test is preferable to any of the above.
Kui plokk on pandud jääle, võetakse see sealt ära ja pannakse tagasi mikrotoomi kelgule.
If t-tests or Dunnett's test is used, there is no need for a ANOVA significance F-test in order to proceed.
b.
For details see the flow chart in Appendix 8.
Mikrotoomi lõigete paksuseks seatakse 3–5 mikronit, kelku liigutatakse edasi mikrotoomi ratta keeramisega.
Results should be reported in tables as concentration means ± SD for males, females, intersex and undifferentiated separately.
Plokist tehakse lõikeid, kuni saadakse riba, milles on vähemalt üks korralik sugunäärmeid hõlmav lõige.
Statistical significance for phenotypic females and phenotypic males should be highlighted.
(Lõikamise puhul võib olla vajalik võtta plokk kelgult, panna koe niisutamiseks jääle ja panna siis tagasi kelgule.)
Test chemical actual concentrations
c.
The actual chamber concentrations of the test chemical should be analysed in frequencies described in paragraph 34.
Lõiked pannakse vesivanni veepinnale lapiti ujuma.
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.
Püütakse leida vähemalt üks lõige, millel ei oleks kortse ja mille all ei oleks kinnijäänud õhumulle.
Examples can be found in the FSDT Phase 2 validation report (42).
d.
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.
Mikroskoobi objektiklaas pannakse vette parima lõike alla ja tõstetakse see klaasi abil veest välja.
Relevant physical-chemical properties;
See protsess on paigaldamine objektiklaasile.
chemical identification data including purity and analytical method for quantification of the test chemical.
e.
Test procedure used (e.g. flow-through semi-static/renewal);
Ühe kala komplekti jaoks valmistatakse kolm lõiget.
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);
Teine ja kolmas lõige tehakse pärast esimest lõiget 50 mikroniliste vahemaade järel.
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;
Kui kala ei ole sisestatud nii, et sugunäärmed oleksid ühes ja samas lõiketasapinnas, tuleb teha rohkem lõikeid; iga kala kohta tuleb saada vähemalt kuus sugunäärmeid hõlmavat lõiget.
Water quality within test chambers:
f.
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.
Markeriga märgitakse objektiklaasile ploki number, millest saadi klaasil olev lõige.
Evidence that controls met the validity criteria:
g.
data on hatching rate should be presented in tables as percentage per replicate and per concentration.
Objektiklaas pannakse värvimisraamile.
Outliers from the acceptance criteria (in controls) should be highlighted.
h.
Survival should be presented as percentage per replicate and per concentration.
Plokk võetakse kelgult ja pannakse hoiukohta esiküljega allapoole.
Outliers from the validity criteria (in controls) should be highlighted;
Värvimine, kaanetamine ja preparaadi märgistamine
Clear indication of the results obtained on the different endpoints observed:
Värvida lõiked histopatoloogiliseks uuringuks.
embryo survival and hatching success;
Püsivalt sulgeda objektiklaasile paigutatud ja värvitud kude.
external abnormalities;
Püsivalt märgistada värvitud lõiked nii, et oleks tagatud täielik jälgitavus.
length and weight;
Värvimine
VTG measurements (ng/g homogenate, ng/ml plasma or ng/mg liver);
a.
gonadal histology, sex ratio, genetic sex data;
Objektiklaase kuivatatakse õhu käes enne värvimist üks öö.
incidence of any unusual reactions by the fish and any visible effects produced by the test chemical.
b.
The results should be presented as mean values ± standard deviation (SD) or standard error (SE).
Lõiked värvitakse hematoksüliini ja eosiiniga.
Statistics should be reported as a minimum as NOEC and LOEC and confidence intervals.
Kaanetamine
The statistical flow chart (Appendix 8) should be followed.
a.
Sumpter, 1996, “Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals”, Environmental Toxicology and Chemistry 15, pp. 194-202.
Kaaned võib peale panna käsitsi või automaadiga.
Sumpter, J.P. and S. Jobling, 1995, “Vitellogenesis As A Biomarker for Estrogenic Contamination of the Aquatic Environment”, Environmental Health Perspectives 103, pp. 173-178.
b.
Hutchinson, S. Maddix, and H. Trip (1999), “An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin”, Environmental Toxicology and Chemistry 18, pp. 337-347.
Objektiklaas kastetakse ksüleeni või TissueClear'i sisse ja liigne vedelik eemaldatakse klaasi ettevaatliku koputamisega.
Pedersen, and P. Bjerregaard (2001a), “Development of an ELISA for vitellogenin in whole body homogenate of zebrafish (Danio rerio)”, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 130, pp. 119-131.
c.
Andersen, L., P. Bjerregaard, and B. Korsgaard (2003), “Vitellogenin induction and brain aromatase activity in adult male and female zebrafish exposed to endocrine disrupters”, Fish Physiology and Biochemistry 28, pp. 319-321.
Objektiklaasi selle serva lähedale, mis on mati ääre vastasküljel, või kaanele kantakse ligikaudu 0,1 ml kinnituskeskkonda.
Petersen (2003), “Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone”, Aquatic Toxicology 65, pp. 397-411.
d.
Tyler (2002), “Utility of a juvenile fathead minnow screening assay for detecting (anti-)estrogenic substances”, Environmental Toxicology and Chemistry 21, pp. 319-326.
Kaant objektiklaasi suhtes väikse nurga all hoides pannakse kaas objektiklaasi peale.
Wang (2007), “Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults”, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 145, pp. 533-541.
Märgistamine
LeBlanc, and C.V.Sullivan (1999), “Fathead minnow (Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds”, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 123, pp. 113-125.
a.
Porcher (2002), “Development and validation of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio rerio)”, Environmental Toxicology and Chemistry 21, pp. 1699-1708.
Igal objektiklaasil esitatakse järgmine teave: