natgillin/translations-raw huggingface dataset:
Hvis der observeres en afvigelse fra testgodkendelseskriterierne, skal følgerne betragtes i forhold til pålideligheden af testdataene, og disse betragtninger inkluderes i rapporten.
Spiking the overlying water
Testbeholderne
Twenty-four hours after adding the first instar larvae for the 1st generation, the test chemical is spiked into the overlying water column, and slight aeration is again supplied (for possible amendments of the test design, see paragraph 7).
Enhver type glas, rustfrit stål eller andet kemisk inaktivt materiale kan anvendes.
Small volumes of the test chemical stock solutions are applied below the surface of the water using a pipette.
Testbeholdernes dimensioner skal være store nok til at opfylde nedenstående kriterier for belastningsgraden.
The overlying water should then be mixed with care not to disturb the sediment.
Det anbefales at placere testbeholderne tilfældigt.
In the spiked water design, exposure starts with the spiking of the water (i.e. one day after addition of the larvae).
Et randomiseret design med grupper af testbeholdere, hvor hver koncentration forefindes i hver gruppe, bør foretrækkes frem for et helt randomiseret system.
Collecting emerged adults
Testbeholderne bør skånes for unødvendige forstyrrelser.
Emerged midges of the 1st generation are collected at least once, but preferably twice a day (see point 36) from the test vessels using an aspirator, exhauster or similar device (see Appendix 5).
Anbefalede fiskearter er anført i tillæg 2.
Special care should be taken not to damage the adults.
Procedurerne for inddragelse af nye arter er anført i punkt 2.
The collected midges from four test vessels within one treatment are released into a breeding cage to which they had been previously assigned.
Opbevaring af forældrefisk
At the day of first (male) emergence, crystallising dishes are spiked by pipetting a small volume of the test chemical stock solution below the water surface (spiked water design).
Oplysninger om opbevaring af forældrefisk under tilfredsstillende omstændigheder kan findes i OECD TG 210 (1).
The concentration of test chemical in the crystallising dish is nominally the same as in the treatment vessels which are assigned to that specific breeding cage.
Forældrefisk fodres en eller to gange om dagen med passende foder.
For the spiked sediment design, the crystallising dishes are prepared at around day 11 after the start of the exposure (i.e. addition of the 1st generation larvae) so that they can equilibrate for about 48 hours before the first egg ropes are produced.
Håndtering af embryoner og fiskelarver
Egg ropes are collected from the crystallising dish in the breeding cage using tweezers or a blunt pipette.
Indledningsvis kan embryoner og fiskelarver, mens de eksponeres for testkemikaliet, opholde sig i mindre i kar af glas eller rustfrit stål med netvægge, idet disse mindre kar er placeret i testkarret, således at testkemikaliet kan flyde igennem.
Each egg rope is placed into a vessel containing culture medium from the crystallising dish it was collected from (e.g. a well of a 12-well micro-plate together with at least 2,5 ml of medium).
Man kan skabe et ikketurbulent flow gennem de mindre kar ved at lade dem hænge ned fra en arm, som bevæger karret op og ned, dog altid med organismerne under overfladen.
The vessels with the egg ropes are covered with a lid to prevent significant evaporation.
I de tilfælde, hvor man har anvendt beholdere, riste eller netværk til at holde æg inde i testkarret, skal disse fjernes, når æggene klækkes, idet man dog beholder det netværk, som hindrer fiskene i at forsvinde.
Egg ropes are kept for observation for at least six days after they have been produced so that they can be classified as fertile or infertile.
Hvis det er nødvendigt at flytte fiskelarver, må de ikke eksponeres for luft, og net må ikke bruges til at fjerne fisk fra beholderne.
For starting the 2nd generation, at least three but preferably six fertile egg ropes are selected from each breeding cage and together with some food allowed to hatch.
Tidspunktet for en sådan overførsel varierer med arten og behøves ikke i alle tilfælde.
These egg ropes should have been produced at the peak of oviposition, which normally occurs around test day 19 in the controls.
Som testvand kan anvendes vand af enhver beskaffenhed, hvori fiskearten i kontrolbeholderne har en overlevelse, der er mindst lige så god som beskrevet i tillæg 3.
Ideally, the 2nd generation of all treatments is initiated on the same day, but due to chemical related effects on larval development, this may not always be possible.
For at sikre, at fortyndingsvandet ikke får urimelig indflydelse på testresultatet (f.eks. ved at reagere med testkemikaliet) eller ved at indvirke uheldigt på de præstationer, man forventer af fisk til yngel, bør man regelmæssigt udtage prøver til analyse.
In such a case, the higher concentrations may be initiated later than the lower treatments and the (solvent) control.
Målinger af totalt organisk kulstof, ledningsevne, pH og opslæmmet tørstof foretages f.eks. hver tredje måned, såfremt man ved, at fortyndingsvandet har en relativt konstant kvalitet.
a.
Målinger af tungmetaller (f.eks. Cu, Pb, Zn, Hg, Cd og Ni), de vigtigste anioner og kationer (f.eks. Ca2+, Mg2+, Na+, K+, Cl–, SO42–) og pesticider foretages, hvis vandkvaliteten er tvivlsom.
In the spiked water design, the sediment-water system for the 2nd generation is prepared by spiking the test chemical into the overlying water column ca.
Nærmere oplysninger om kemisk analyse og vandopsamling kan ses i punkt 34.
1 hour before adding the first instar larvae to the test vessels.
Et gennemstrømningssystem bruges, hvis det er praktisk muligt.
Small volumes of the test chemical solutions are applied below the surface of the water using a pipette.
Til gennemstrømningstest kræves et system, som kontinuerligt doserer og fortynder en stamopløsning af testkemikaliet (f.eks. udmålingspumpe, proportionalfortynder, mætningssystem), så der afgives en række forskellige koncentrationer til testbeholderne.
After spiking, slight aeration is supplied.
Stamopløsningernes og fortyndingsvandets strømningshastigheder kontrolleres regelmæssigt i løbet af testen og må ikke variere mere end 10 % gennem hele testforløbet.
b.
En flow-hastighed svarende til volumenet af mindst fem målekamre pr. døgn er fundet passende (1).
In the spiked sediment design, the exposure vessels containing the sediment-water system for the 2nd generation are prepared in the same way as for the 1st generation.
Der må udvises forsigtighed, så brugen af plastslanger eller andre materialer undgås, da nogle af dem kan indeholde biologisk aktive kemikalier eller kan adsorbere testkemikaliet.
Twenty first instar larvae (maximum 48 h post hatching) of the 2nd generation are allocated randomly to each test vessel containing the spiked sediment-water system, using a blunt pipette.
Stamopløsningen fremstilles fortrinsvis uden brug af opløsningsmidler, men ved mekanisk opblanding eller omrystning af testkemikaliet i fortyndingsvandet (f.eks. ved omrøring eller ultralydbehandling).
Aeration of the water should be stopped while adding the first instar larvae to the test vessels and remain so for another 24 hours after addition of the larvae.
Hvis testkemikaliet er vanskeligt at opløse i vandet, følges procedurerne i OECD's Guidance Document on aquatic toxicity testing of difficult substances and mixtures (36).
According to the test design used (see paragraphs 19 and 20), the number of larvae used per concentration is at least 120 (6 replicates per concentration) for the ECX approach and 160 for the NOEC approach (8 replicates per concentration).
Anvendelse af opløsningsmidler bør undgås, men kan i nogle tilfælde være nødvendig for at fremstille en stamopløsning med en passende koncentration.
It is necessary to feed the larvae in the test vessels, preferably daily or at least three times per week.
Eksempler på egnede opløsningsmidler er anført i (36).
Fish-food (a suspension in water or finely ground food, e.g. Tetra-Min or Tetra-Phyll; see details in Appendix 2) of 0,25 - 0,5 mg (0,35 - 0,5 mg for C. yoshimatsui) per larvae per day is an adequate amount of food for young larvae during the first 10 days of their development.
Semistatiske testbetingelser bør undgås, medmindre det godtgøres, at der er tvingende årsager hertil i forbindelse med testkemikaliet (f.eks. stabilitet, begrænset tilgængelighed, høj pris eller fare).
Slightly more food may be necessary for older larvae:
Eksponeringsbetingelser
0,5 - 1,0 mg per larvae per day should be sufficient for the rest of the test.
Udtagning af æg og varighed
The food ration should be reduced in all treatments and control if fungal growth is seen or if mortality is observed in controls.
For at undgå genetisk skævhed udtages æg fra mindst tre blandede og tilfældigt udvalgte ynglende par eller grupper som indledning på testen.
If fungal development cannot be stopped the test should be repeated.
Se beskrivelse af kunstig befrugtning for trepigget hundestejle i tillæg 11.
The toxicological relevance of exposure via ingestion is generally higher in chemicals with a high affinity for organic carbon or chemicals covalently binding to the sediment.
Testen påbegyndes snarest muligt, efter at æggene er blevet befrugtet, idet embryonerne helst skal være nedsænket i testopløsningerne, før eller så snart som muligt efter at blastocysten har delt sig og senest 12 timer efter befrugtning.
Hence, when testing chemicals with such properties, the amount of food necessary to ensure survival and natural growth of the larvae may be added to the formulated sediment before the stabilisation period, depending on the regulatory demand.
Testen fortsættes, indtil kønsdifferentiering i kontrolgruppen er afsluttet (60 dage efter udklækning for japansk risfisk, trepigget hundestejle og zebrafisk).
To prevent deterioration of the water quality, plant material should be used instead of fish food, e.g. addition of 0,5 % (dry weight) finely ground leaves of stinging nettle (Urtica dioica), mulberry (Morus alba), white clover (Trifolium repens), spinach (Spinacia oleracea) or other plant material (Cerophyl or α-cellulose).
Belastning
Addition of the complete ration of an organic food source to the sediment before spiking is not trivial with respect to water quality and biological performance (21), nor a standardised method, but recent studies provide indications that this method works (19) (26).
Antallet af befrugtede æg ved testens begyndelse skal være mindst 120 pr. koncentration fordelt på mindst fire replikater (fordeling til kontrol ifølge kvadratrodsreglen accepteres).
Adult midges in the breeding cage need no feeding normally, but fecundity and fertility are enhanced when a cotton wool pad soaked in a saturated sucrose solution is offered as a food source for emerged adults (34).
Æggene fordeles tilfældigt (ved hjælp af statistiske tabeller for randomisering) mellem behandlingerne.
Gentle aeration of the overlying water in the test vessels is supplied 24 hours after addition of the first instar larvae of both generations and is continued throughout the test (care should be taken that the dissolved oxygen concentration does not fall below 60 % of ASV).
Belastningsgraden (se definition i tillæg 1) bør være så lav, at koncentrationen af opløst ilt på mindst 60 % ASV kan opretholdes uden direkte beluftning af beholderne.
Aeration is provided through a glass Pasteur pipette of which the outlet is fixed 2-3 cm above the sediment layer giving a few bubbles/sec.
Hvis der forekommer dødelighed som følge af eksponering, reduceres antallet af replikater i overensstemmelse hermed, så fisketætheden holdes så lige som muligt mellem behandlingsniveauer.
When testing volatile chemicals, consideration should be given not to aerate the sediment-water system, while at the same time the validity criterion of minimal 60 % ASV (paragraph 10) should be fulfilled.
Lysperioderne og vandtemperaturen skal passe til den art, der bruges i testen (se forsøgsbetingelser for FSDT i tillæg 2).
Further guidance is provided in (16).
Foder og fodring er afgørende, og det er væsentligt, at der gives det korrekte foder til hvert stadium med passende tidsintervaller og i en tilstrækkelig mængde til at støtte normal vækst.
The test with C. riparius is conducted at a constant temperature of 20 °C (± 2 °C).
Foder gives ad libitum, samtidig med at overskydende foder undgås.
For C. dilutus and C. yoshimatsui, recommended temperatures are 23 °C and 25 °C (± 2 °C), respectively.
For at sikre en tilstrækkelig vækstrate fodres fiskene mindst to gange om dagen (dog accepteres en gang om dagen i weekender) med et interval på mindst tre timer mellem hver fodring.
A 16 hours photoperiod is used and the light intensity should be 500 to 1000 lux.
Overskydende foder og ekskrementer fjernes efter behov for at undgå akkumulering af affald.
For the breeding cages an additional one hour dawn and dusk phase may be included.
Foder og fodringshyppighed justeres til stadighed, i takt med at der indhøstes erfaringer, for at forbedre overlevelse og optimere vækst.
Exposure duration
Det bør derfor tilstræbes at bekræfte den foreslåede hyppighed med anerkendte eksperter.
Spiked water design:
Der fodres ikke 24 timer før testens afslutning.
The exposure period of the 1st generation starts when the test chemical is spiked into the overlying water of the test vessels (which is one day after insertion of the larvae — for possible amendments of the exposure design, see paragraph 7).
Eksempler på passende fødemidler er anført i tillæg 2 (se også OECD's Fish Testing Framework (39)).
Exposure of the 2nd larval generation starts immediately, since they are inserted into a sediment-water system that has been already spiked.
Kurven fra de tilgængelige akutte toksicitetstest, som knytter LC50 til testperioden, bør tages i betragtning ved valg af området for testkoncentrationerne.
For C. dilutus, maximum exposure durations are 64 and 65 days, for the 1st and 2nd generation, respectively.
Fem testkoncentrationer anbefales, hvis dataene skal bruges til risikovurdering.
The total duration is approximately 100 days.
Det er ikke nødvendigt at teste kemikaliekoncentrationer højere end svarende til 10 % af akutte voksne LC50 eller 10 mg/l, afhængigt af hvilken af disse der er lavest.
Spiked sediment design:
Den maksimale testkoncentration bør være 10 % af LC50 på larve-/yngelstadiet.
exposure starts with the addition of the larvae and is maximum 28 days for both generations for C. riparius and C. yoshimatsui and maximum 65 days for both generations for C. dilutus.
Kontrol
Emergence
Der skal ud over testkoncentrationerne være en kontrol med fortyndingsvand (≥ 4 replikater) og, hvis det er relevant, en kontrol med opløsningsmiddel (≥ 4 replikater).
Development time and the total number of fully emerged and alive male and female midges are determined for both generations.
Kun opløsningsmidler, der er blevet undersøgt og har vist, at de ikke har nogen statistisk signifikant indflydelse på testens endepunkter, bør anvendes i testen.
Males are easily identified by their plumose antennae and thin body posture.
Hvis der anvendes et opløsningsmiddel, må dets endelige koncentration ikke være højere end 0,1 ml/l (36), og det skal være den samme koncentration i alle testbeholdere med undtagelse af kontrollen med fortyndingsvand.
Test vessels of both generations should be observed at least three times per week to make visual assessment of any abnormal behaviour of the larvae (e.g. leaving sediment, unusual swimming), compared to the control.
Det bør dog så vidt muligt undgås at bruge sådanne opløsningsmidler, ellers holdes opløsningsmidlets koncentrationer på et minimum.
During the period of emergence, which starts about 12 days after insertion of the larvae for C. riparius and C. yoshimatsui (after 20 days for C. dilutus), emerged midges are counted and sexed at least once, but preferably twice a day (early morning and late afternoon).
Hyppighed af analyser og målinger
After identification, the midges of the 1st generation are carefully removed from the vessels and transferred to a breeding cage.
Der udføres en kemisk analyse af testkemikaliets koncentration før indledning af testen for at kontrollere, at den opfylder godkendelseskriterierne.
Midges of the 2nd generation are removed and killed after identification.
Alle replikater analyseres hver for sig ved påbegyndelse og afslutning af testen.
Any egg ropes deposited in the test vessels of the 1st generation should be collected individually and transferred with at least 2,5 ml native water to 12-well microplates (or other suitable vessels) which are covered with a lid to prevent significant evaporation.
Et replikat pr. testkoncentration analyseres mindst en gang om ugen under testen, idet der systematisk veksles mellem replikaterne (1,2,3,4,1,2 osv.).
The number of dead larvae and visible pupae that have failed to emerge should also be recorded.
Opbevares prøver med henblik på analyse på et senere tidspunkt, valideres opbevaringsmetoden for prøverne på forhånd.
Examples of a breeding cage, test vessel and exhauster are provided in Appendix 5.
Prøverne filtreres (f.eks. med et filter med en porestørrelse på 0,45 μm) eller centrifugeres for at sikre, at bestemmelserne foretages på testkemikaliet i opløsning.
Reproduction
Under testen måles opløst ilt, pH, samlet hårdhed, ledningsevne, saltindhold (hvis det er relevant) og temperatur i alle testbeholdere.
Effects on reproduction are assessed via the number of egg ropes produced by the 1st generation of midges and the fertility of these egg ropes.
Som minimum måles opløst ilt, saltindhold (hvis det er relevant) og temperatur en gang om ugen og pH, ledningsevne og hårdhed ved testens begyndelse og afslutning.
Once per day the egg ropes are collected from the crystallising dish that is placed in each breeding container.
Temperaturen overvåges fortrinsvis kontinuerligt i mindst en testbeholder.
The egg ropes should be collected and transferred with at least 2,5 ml native water to a 12-wells microplate (one egg rope in each well) or other suitable vessels, which are covered with a lid to prevent significant evaporation.
Resultaterne baseres på målte koncentrationer.
The following characteristics are documented for each egg rope:
Hvis testkemikaliets koncentration i tilstrækkelig grad har holdt sig inden for ± 20 % af den nominelle koncentration gennem hele testen, kan resultaterne baseres på enten de nominelle eller de målte værdier.
day of production, size (normal, i.e. 1,0 ± 0,3 cm or small; typically ≤ 0,5 cm), and structure (normal = banana-form with spiralled egg string or abnormal, e.g. unspiralled egg string) and fertility (fertile or infertile).
Observationer og målinger
Over the course of six days after it was produced the fertility of an egg rope is assessed.
Fosterets udviklingstrin
An egg rope is considered fertile when at least one third of the eggs hatch.
Eksponeringen påbegyndes så hurtigt som muligt efter befrugtning, og før blastocysten har delt sig, og senest 12 timer efter befrugtning for at sikre eksponering under den tidlige fosterudvikling.
The total number of females added to the breeding cage is used to calculate the number of egg ropes per female and the number of fertile egg ropes per female.
Udklækning og overlevelse
If required, the number of eggs in an egg rope can be estimated non-destructively by using the ring count method (detailed in 32 and 33).
Udklækning og overlevelse observeres mindst en gang om dagen og noteres.
Analytical measurements
Døde embryoner, larver og fiskeyngel fjernes med det samme, eftersom de hurtigt kan gå i opløsning og blive bidt i stykker af de øvrige fisk.
Concentration of the test chemical
Meget stor omhu skal udvises, når døde individer fjernes, så nærtliggende æg eller larver ikke bliver ramt eller fysisk beskadigede, da de er meget sarte og følsomme.
As a minimum, samples of the overlying water, pore water and the sediment should be analysed at the start of exposure (in case of water spiking preferably one hour after application) and at the end of the test, at the highest concentration and a lower one.
Kriterier for død varierer efter stadium:
This applies to vessels from both generations.
for æg:
From the crystallising dishes in the breeding cage only the overlying water is analysed, since this is what the egg ropes come into contact with (for the spiked sediment design an analytical confirmation of the sediment concentration may be considered).
særligt i de tidlige stadier et tydeligt tab af gennemsigtighed og ændring i farve forårsaget af koagulation og/eller bundfældelse af proteiner, hvilket medfører en hvid uklarhed
Further measurements of sediment, pore water or overlying water during the test may be conducted if deemed necessary.
for larver og fiskeyngel:
These determinations of test chemical concentration inform on the behaviour/partitioning of the test chemical in the water-sediment system.
manglende bevægelighed og/eller manglende respirationsbevægelser og/eller manglende hjerteslag og/eller hvid uklar farvning af centralnervesystemet og/eller manglende reaktion på mekaniske stimuli.
Sampling of sediment and pore water at the start and during the test (see paragraph 39) requires additional test vessels to perform analytical determinations.
Abnormt udseende
Measurements in sediment in the spiked water design might not be necessary if the partitioning of the test chemical between water and sediment has been clearly determined in a water/sediment study under comparable conditions (e.g. sediment to water ratio, type of application, organic carbon content of sediment), or if measured concentrations in the overlying water are shown to remain within 80 to 120 % of the nominal or measured initial concentrations..
Antallet af larver eller fisk med abnorm kropsform registreres, og abnormitetens udseende og art beskrives.
When intermediate measurements are made (e.g. at day 7 and/or 14) and if the analysis needs large samples which cannot be taken from test vessels without influencing the test system, analytical determinations should be performed on samples from additional test vessels treated in the same way (including the presence of test organisms) but not used for biological observations.
Det skal bemærkes, at abnorme embryoner og larver optræder almindeligt og kan opgøres til adskillige procent i kontrollerne hos visse arter.
Centrifugation at e.g. 10000 g at 4 °C for 30 min is the recommended procedure to isolate interstitial (= pore) water.
Abnorm adfærd
Physical-chemical parameters
Abnorm adfærd, f.eks. hyperventilation, ukoordineret svømning, atypisk ubevægelighed og atypisk fourageringsadfærd, noteres når det forekommer.
pH, dissolved oxygen in the test water and temperature of the water in the test vessels and crystallising dishes should be measured in an appropriate manner (see paragraph 10).
Vægt
Hardness and ammonia should be measured in the controls and in one test vessel and crystallising dish at the highest concentration at the start and the end of the test.
Ved testens afslutning aflives alle overlevende fisk (bedøves, hvis der skal tages blodprøver), og individuel vådvægt (duppet tør) måles.
The purpose of this life-cycle test is to determine the effect of the test chemical on the reproduction and, for two generations, the development rate and the total number of fully emerged and alive male and female midges.
Længde
For the emergence ratio data of males and females should be pooled.
Ved testens afslutning måles de enkelte individers længde (standardlængde).
If there are no statistically significant differences between the sensitivities in the development rate of the separate sexes, male and female results may be pooled for statistical analysis.
Disse observationer vil medføre, at nogle eller alle nedenstående data kan registreres:
Effect concentrations expressed as concentrations in the overlaying water (for spiked water) or in the sediment (for spiked sediment), are usually calculated based on measured concentrations at the beginning of the exposure (see paragraph 38).