dapthoxkit f magna test procedure pdf...21 shake the vial with the spirulina suspension, pour it in...
TRANSCRIPT
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DAPTHOXKIT F MAGNA
Test procedure
![Page 2: DAPTHOXKIT F MAGNA Test procedure PDF...21 SHAKE THE VIAL WITH THE SPIRULINA SUSPENSION, POUR IT IN THE PETRI DISH WITH THE DAPHNIA NEONATES AND SWIRL THE PETRI DISH GENTLY 26 TO AVOID](https://reader033.vdocument.in/reader033/viewer/2022050508/5f9909a806f58b18f649d7a8/html5/thumbnails/2.jpg)
1
PREPARATION OF
STANDARD FRESHWATER
- VOLUMETRIC FLASK (2 liter)
- VIALS WITH SOLUTIONS OF
CONCENTRATED SALTS
- DISTILLED (or deionized) WATER
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2
POUR THE 4 VIALS
WITH CONCENTRATED SALT SOLUTIONS
IN + 1 LITER DISTILLED WATER,
IN THE 2 LITER VOLUMETRIC FLASK
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3
FILL THE FLASK TO THE 2 LITER MARK
AND AERATE FOR AT LEAST 15 MINUTES
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4
HATCHING OF THE EPHIPPIA
REMOVE THE
ALUMINIUM FOIL
FROM A TUBE
WITH DAPHNIA
EPHIPPIA
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5
POUR THE CONTENTS
OF THE TUBE WITH EPHIPPIA
IN THE MICROSIEVE
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6
MAKE SURE THAT
ALL THE EPHIPPIA
ARE TRANSFERRED
INTO THE MICROSIEVE
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7
RINSE THE EPHIPPIA
THOROUGHLY
WITH TAP WATER
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8
TRANSFER THE EPHIPPIA
INTO THE
HATCHING PETRI DISH
IN STANDARD FRESHWATER
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9
INCUBATE THE PETRI DISH
FOR 72h AT 20-22 °C
UNDER CONTINUOUS ILLUMINATION
OF 6 000 LUX
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10
PREPARATION OF THE
TOXICANT DILUTIONS
For example :
TEST ON A EFFLUENT
IN 5 DILUTIONS (C1-C5)
+ ONE CONTROL
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11
TRANSFER 50 ML
STANDARD FRESHWATER
INTO FLASKS
C2, C3, C4 AND C5
50 ml
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12
FILL FLASK C1
TO THE 100 ML MARK
WITH EFFLUENT
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13
TRANSFER 50 ML EFFLUENT
FROM FLASK C1
INTO A GRADUATED CYLINDER.
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14
TRANSFER THE 50 ML EFFLUENT
FROM THE GRADUATED CYLINDER
TO FLASK C2 AND SHAKE
THOROUGHLY
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15
50 ml50 ml50 ml
REPEAT THE FORMER
DILUTION PROCEDURE
FOR THE OTHER FLASKS
(i.e. 50 ml from C2 to C3, etc).
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16
FILLING OF THE TEST PLATE :
TRANSFER 10 ML
STANDARD FRESHWATER
INTO EACH WELL
OF THE CONTROL ROW
Control row
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17
TRANSFER 10 ML OF THE
RESPECTIVE TOXICANT
CONCENTRATIONS
INTO EACH WELL
OF THE CORRESPONDING ROWS
FROM C5 TO C1
Test rows
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18
AFTER 72h TO 80h
INCUBATION
VERIFY THE HATCHING
OF THE DAPHNIA NEONATES
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19
A MINIMUM OF 120 NEONATES
ARE NEEDED TO PERFORM
ONE TEST AND THE NEONATES
SHOULD NOT BE OLDER THAN 24H
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20
2h PRE-FEEDING
OF THE TEST ORGANISMS
TAKE ONE VIAL
WITH SPIRULINA POWDER
AND FILL IT
WITH STANDARD FRESHWATER
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21
SHAKE THE VIAL
WITH THE SPIRULINA SUSPENSION,
POUR IT IN THE PETRI DISH
WITH THE DAPHNIA NEONATES
AND SWIRL THE PETRI DISH GENTLY
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SET UP
22
OF THE TRANSFER
OF THE DAPHNIAS
TO THE TEST WELLS
- MULTIWELL PLATE
- LIGHT BOX WITH
TRANSPARENT STAGE
- MICROPIPETTE
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23
TRANSFER AT LEAST 20
(actively swimming)
DAPHNIAS INTO
THE RINSING CUP
OF THE CONTROL ROW,
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24
TRANSFER 20 DAPHNIAS (minimum)
TO ALL THE OTHER RINSING CUPS,
IN ORDER OF INCREASING
CONCENTRATIONS OF TOXICANT
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25
TRANSFER EXACTLY 5
DAPHNIAS FROM EACH
RINSING WELL
INTO THE 4 WELLS
OF THE CORRESPONDING
ROW
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26
TO AVOID SURFACE FLOATING
OF THE DAPHNIAS
DURING THE TRANSFER,
PUT THE TIP OF THE
MICROPIPETTE IN THE MEDIUM,
AND DO NOT DROP THE ORGANISMS
AT THE SURFACE OF THE MEDIUM
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27
PUT A PIECE OF PARAFILM
ON THE MULTIWELL PLATE
AND PUT THE COVER
ON TIGHTLY
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28
INCUBATION OF THE TEST PLATE
INCUBATE THE MULTIWELL
AT 20 + 2 °C IN DARKNESS
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29
SCORING OF THE RESULTS
AFTER 24h AND 48h INCUBATION
PUT THE MULTIWELL PLATE
ON THE LIGHT TABLE
AND RECORD THE NUMBER
OF DEAD AND
IMMOBILIZED DAPHNIAS
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30
DAPHNIAS WHICH ARE NOT
ABLE TO SWIM
AFTER GENTLE AGITATION
OF THE LIQUID FOR 15 SECONDS
SHALL BE CONSIDERED
AS IMMOBILIZED
(even if they can still
move their antennae)
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- SCORE THE FIGURES
ON THE RESULTS SHEET.
31
ON THE RESULTS SHEET.
- CALCULATE THE TOTAL NUMBER
OF DEAD AND IMMOBILE DAPHNIAS
FOR EACH TOXICANT
CONCENTRATION
- CALCULATE THE MEAN EFFECT
AND THE PERCENTAGE EFFECT