Sampling, Laboratory Sampling, Laboratory Analysis and Calculation Analysis and Calculation methods for Resource methods for Resource Assessment of ArtemiaAssessment of Artemia

Naser Agh, Naser Agh, RaminRamin ManaffarManaffar, , BehroozBehrooz AtashbarAtashbarArtemia & Aquatic Animals Research Center,Artemia & Aquatic Animals Research Center,

Urmia University, Urmia, IranUrmia University, Urmia, Iran

Urmia LakeUrmia LakeSampling SitesSampling Sites

12 sites in South 12 sites in South 7 Sites in North7 Sites in North

Anchor the boat Anchor the boat Find the coordinates Find the coordinates -- GPS unit. GPS unit.

Measure the depth Measure the depth of the waterof the water

Depth Sounder

1. Collect the sample using a plankton 1. Collect the sample using a plankton net net -- 0.5m from the bottom 0.5m from the bottom 2. Slowly raise the plankton net to the 2. Slowly raise the plankton net to the water surface. water surface. 3. Using a pump or bucket, wash the 3. Using a pump or bucket, wash the outside of the net thoroughly with lake outside of the net thoroughly with lake water to rinse all of the zooplankton water to rinse all of the zooplankton into the sampling cup. into the sampling cup. 4. Allow excess water to drain from 4. Allow excess water to drain from sample cup. sample cup. 5. Remove the sampling cup from the 5. Remove the sampling cup from the net. net. 6. Wash the contents of the cup into a 6. Wash the contents of the cup into a sample bottle using filtered lake water sample bottle using filtered lake water 7. Repeat steps 47. Repeat steps 4--9 and combine with 9 and combine with Artemia in the same sample bottle as Artemia in the same sample bottle as the first net haul.the first net haul.8. Place bottle in a cooler filled with 8. Place bottle in a cooler filled with ice. ice. 9. Same process is repeated for 9. Same process is repeated for collecting samples from 0.5 m from collecting samples from 0.5 m from water surfacewater surface

Alpha BottlesAlpha Bottles

Horizontal

Vertical

Collecting samples for assessment Collecting samples for assessment of phytoplanktonesof phytoplanktones

Studying Lake ChemistryStudying Lake Chemistry

Dissolved Oxygen: Dissolved Oxygen: 1. Calibrate the dissolved oxygen meter 1. Calibrate the dissolved oxygen meter 2. Place the probe into the lake 0.5m below the surface of the w2. Place the probe into the lake 0.5m below the surface of the water. ater. 3. Record the temperature, % saturation and mg/L on the display.3. Record the temperature, % saturation and mg/L on the display.4. Repeat steps 14. Repeat steps 1--3 at 0.5 m from the bottom of the lake. 3 at 0.5 m from the bottom of the lake.

Salinity and Temperature:Salinity and Temperature:1. Collect a sample from the surface of the water in any contain1. Collect a sample from the surface of the water in any container. er. 2. Place the thermometer in container and record the temperature2. Place the thermometer in container and record the temperature. . 3. Use disposable transfer pipette to place a small amount of wa3. Use disposable transfer pipette to place a small amount of water on ter on the refractometer. the refractometer. 4. Record salinity. 4. Record salinity. 5. Repeat steps 15. Repeat steps 1--4 at 0.5 meters from the bottom of the lake (This 4 at 0.5 meters from the bottom of the lake (This water can be collected while determining the depth of the lake fwater can be collected while determining the depth of the lake for use or use of the plankton net). of the plankton net). 6. Wash refractometer thoroughly after6. Wash refractometer thoroughly after use.use.

Water TransparencyWater Transparency: : 1. Lower the 1. Lower the secchisecchi disc into the water until no longer visible. disc into the water until no longer visible. 2. Record that depth while still keeping the disc in the water. 2. Record that depth while still keeping the disc in the water. 3. Raise the disc until visible. 3. Raise the disc until visible. 4. Record that depth. 4. Record that depth. 5. Repeat steps15. Repeat steps1--4 three times. 4 three times.

6. The mean of these depths is the estimated visible depth.6. The mean of these depths is the estimated visible depth.

Sample Preparation:Sample Preparation:separate size/age classes using 850um, 500um, and 125um testing separate size/age classes using 850um, 500um, and 125um testing sieves sieves Carefully run water through the sieves to facilitate complete seCarefully run water through the sieves to facilitate complete separation of size classes. paration of size classes. Rinse water through each sieve separately.Rinse water through each sieve separately.Transfer the biomass contents from the sieve into 3 different seTransfer the biomass contents from the sieve into 3 different settling cones.ttling cones.Increase the volume of liquid in the cone to a known levelIncrease the volume of liquid in the cone to a known levelAerate contents of cone for thorough mixing.Aerate contents of cone for thorough mixing.Pipette 250ul subPipette 250ul sub--samples from the cone (containing biomass from 125ul sieve) intosamples from the cone (containing biomass from 125ul sieve) into each well of a 24each well of a 24--well well microplate. If the cone contains a low density, a higher volumemicroplate. If the cone contains a low density, a higher volume can be introduced into each well for can be introduced into each well for counting.counting.Add one drop of concentrated LugolAdd one drop of concentrated Lugol’’s iodine to each well in the plates iodine to each well in the plateRecord the volume and number of subRecord the volume and number of sub--samples to be counted samples to be counted Pipette 5ml subPipette 5ml sub--samples from the cones containing biomass from 500 & 850 ul sievsamples from the cones containing biomass from 500 & 850 ul sieves into each well of a es into each well of a 66--well microplatewell microplateAdd 2Add 2--3 drops of Lugol3 drops of Lugol’’s iodine to each well and set aside to count when entire sample s iodine to each well and set aside to count when entire sample is prepared.is prepared.Record the volume and number of subRecord the volume and number of sub--samples to be counted.samples to be counted.

Sample EnumerationSample Enumeration

Using a stereomicroscope and multicounter, count Using a stereomicroscope and multicounter, count and record contents of the 24and record contents of the 24--well microplate (the well microplate (the contents of all 24 wells added together).contents of all 24 wells added together).After counting the contents, add 5After counting the contents, add 5--10 drops of 5% 10 drops of 5% sodium hypochlorite (household bleach) and 1 sodium hypochlorite (household bleach) and 1 drop of sodium hydroxide (NaOH) into each well to drop of sodium hydroxide (NaOH) into each well to dissolve the shells in the sample (If NaOH is not dissolve the shells in the sample (If NaOH is not available bleach will work by itself but will take available bleach will work by itself but will take longer to dissolve the shells).longer to dissolve the shells).After about 10 minutes the shells will be dissolved, After about 10 minutes the shells will be dissolved, count and record the remaining full cysts.count and record the remaining full cysts.Count and record the number of individuals of Count and record the number of individuals of each age class in the other 2 each age class in the other 2 microplatesmicroplates. .

333.3333333(9 / (2*3)) * 115Juveniles Collected

58000(8 / (2*3)) * 114Combined Meta and Nauplii Collected

17333.33333(7 / (2*3)) * 113Meta-Nauplii Collected

40666.66667(6 / (2*3)) * 112Nauplii Collected

8666.666667(5 / (2*3)) * 111Full Cysts Collected

33333.33333(4 / (2*3)) * 110Spheres Collected

29Juvenile Count

3486 + 78Combined Meta and Nauplii Count

1047Meta-Nauplii Count

2446Nauplii Count

525Full Cyst Count

2004Sphere Count

243125 um Sieve Count Replicates

0.252125 um Sieve Count Volume ml

10001125 um Sieve Volume ml

270(23 / (17*18)) * 1628Pre-adults Collected

7740(22 / (17*18)) * 1627Juveniles Collected

22.5(21 / (17*18)) * 1626Combined Meta and Nauplii Collected

0(20 / (17*18)) * 1625Meta-Nauplii Collected

22.5(19 / (17*18)) * 1624Nauplii Collected

1223Pre-adult Count

34422Juvenile Count

119 + 2021Combined Meta and Nauplii Count

020Meta-Nauplii Count

119Nauplii Count

418500 um Sieve Count Replicates

517500 um Sieve Count Volume

45016500 um Sieve Volume ml

155.5555556(43 / (30*31)) * 2955Gravid Females Collected

0(42 / (30*31)) * 2954Ovi/Ova Females Collected

0(41 / (30*31)) * 2953Females with Nauplii Collected

133.3333333(40 / (30*31)) * 2952Females with Cysts Collected

22.22222222(39 / (30*31)) * 2951Females with Embryos Collected

933.3333333(38 / (30*31)) * 2950Females Collected

866.6666667(37 / (30*31)) * 2949Males Collected

2666.666667(36 / (30*31)) * 2948Pre-adults Collected

0(35 / (30*31)) * 2947Juveniles Collected

0(34 / (30*31)) * 2946Combined Meta and Nauplii Collected

0(33 / (30*31)) * 2945Meta-Nauplii Collected

0(32 / (30*31)) * 2944Nauplii Collected

739 + 40 + 41 + 4243Gravid Females

042Ovi/Ova Females

041Females with Nauplii

640Females with cysts

139Females with embryos

4238Adult female Counts

3937Adult male Counts

12036Pre-adult Count

035Juvenile Count

032 + 3334Combined Meta and Nauplii Count

033Meta-Nauplii Count

032Nauplii Count

931850 um Sieve Count Replicates

530850 um Sieve Count Volume ml

100029850 um Sieve Volume ml

51522 03957M + 0 + S + U + W + YZTOTAL INDIVIDUALS / m3

678.8866259X / KYFemales / m3

933.333333350XFemales

630.3947241V / KWMales / m3

866.666666749VMales

2136.068277T / KUPre-adults / m3

2936.66666728 + 48TPre-adults

5872.369314R / KSJuveniles / m3

8073.33333315 + 27 + 47RJuveniles

42204.32063P / KQMeta-nauplii +Nauplii / m3

58022.514 + 26 + 46PCombined Nauplii and Meta

12607.89448N / KOMeta / m3

17333.3333313 + 25 + 45NMeta-Nauplii

29596.42615L / KMNauplii / m3

40689.1666712 + 24 + 44LNauplii

1.3748I * J * HKVolume M3

3.5JDepth M

0.1964INet Surface M2

2HNet Hauls

Plankton NetMethod

3.231 mGSecchi M

261.0 pptFSalinity Surface PPT

268.0 pptESalinity Bottom PPT

21.30CDTemp Surface C

21.1oCCTemp Bottom C

N:40.52.36, W:112.14.07BCoordinates

May 31 2001ADate

ExampleEquationCode

26.00%(XX / W) * 100AAA% Full Cysts

17942.00369YY / KZZShells / m3

24666.66667UU - WWYYShells

6303.947241WW / KXXFull Cysts / m3

8666.66666711WWFull Cysts

24245.95093UU / KVVSpheres / m3

33333.3333310UUSpheres

0LL * SSTTPotential Nauplii / m3

14From brood sack countsSSAverage Nauplii / Female

376.6204377FF * QQRRPotential Eggs / m3

23.3From brood sack countsQQAverage Eggs / Female

2618.5627JJ * OOPPPotential Cysts / m3

27From brood sack countsOOAverage Cysts / Female

16.67%BB / YNN% Gravid Females

14.29%JJ / YMM% Gravid with Cysts

0KK / KLLFemales with Nauplii / m3

053KKFemales with Nauplii

96.9838037II / KJJFemales with Cysts / m3

133.333333352IIFemales with Cysts

0GG / K HHOvi-Ova Females / m3

054GGOvi-Ova Females

16.16396728EE / KFFFemales with eggs / m3

22.2222222251EEFemales with eggs

565.7388549CC / K DDEmpty Females / m3

777.777777850 - 55CCEmpty Females

113.147771AA / KBBGravid Females / m3

155.555555655AAGravid Females

Brood Size EnumerationBrood Size EnumerationRandomly locate 10 females with brood sacs containing Randomly locate 10 females with brood sacs containing cysts, 10 with embryos, and 10 with nauplii from the adult cysts, 10 with embryos, and 10 with nauplii from the adult fraction of the sample (if less than 10 are available just fraction of the sample (if less than 10 are available just count what is there). count what is there). Place females with brood sacks on a flat surface (a Place females with brood sacks on a flat surface (a petripetridish or microscope slide works well), each female being dish or microscope slide works well), each female being separated by space to avoid brood sack contents from separated by space to avoid brood sack contents from mixing together.mixing together.Place 1 drop of water onto each female so they do not Place 1 drop of water onto each female so they do not become desiccated.become desiccated.Stain each female with 1 drop of LugolStain each female with 1 drop of Lugol’’s iodine for easier s iodine for easier counting.counting.With the aide of a stereomicroscope, tease apart the brood With the aide of a stereomicroscope, tease apart the brood sac to expose all of the contents. This can be done using sac to expose all of the contents. This can be done using a small razor blade, fine tweezers or a dissection probe.a small razor blade, fine tweezers or a dissection probe.Count and record the number of embryos, nauplii or cysts Count and record the number of embryos, nauplii or cysts each female has in her brood sackeach female has in her brood sackCalculate the average number of nauplii, average number Calculate the average number of nauplii, average number of cysts, and average number of embryos per brood sack of cysts, and average number of embryos per brood sack for the sampling location.for the sampling location.

Biomass EstimationBiomass EstimationCombine the contents of the 3 Combine the contents of the 3 ImhoffImhoff settling cones by pouring them through settling cones by pouring them through the 125um testing sieve.the 125um testing sieve.Carefully rinse this mixture back into one of the cones.Carefully rinse this mixture back into one of the cones.Add just enough liquid to allow adequate mixing with aeration anAdd just enough liquid to allow adequate mixing with aeration and record the d record the volume in the cone.volume in the cone.Aerate sample for thorough mixing.Aerate sample for thorough mixing.Record the weight of one filter paper and place it into the Record the weight of one filter paper and place it into the BuchnerBuchner funnel funnel attached to filtering flask and vacuum pump.attached to filtering flask and vacuum pump.Turn vacuum pump on and run a small amount of water through filtTurn vacuum pump on and run a small amount of water through filter to er to establish the vacuum pressure. (The wet filter paper must coverestablish the vacuum pressure. (The wet filter paper must cover the entire the entire funnel in order avoid loss of biomass and to create enough pressfunnel in order avoid loss of biomass and to create enough pressure to filter the ure to filter the water out.)water out.)Using pipette with the 10ml attachment, introduce the biomass miUsing pipette with the 10ml attachment, introduce the biomass mixture from the xture from the cone into the cone into the BuchnerBuchner funnel.funnel.When no more water can be vacuumed from the sample turn off the When no more water can be vacuumed from the sample turn off the pump and pump and remove the filter with attached biomass.remove the filter with attached biomass.Weigh the filter paper plus biomass then record the Weigh the filter paper plus biomass then record the ““wet weightwet weight”” of this sample. of this sample. A A ““wet weightwet weight”” of 5g is desired to avoid problems when trying to dry the of 5g is desired to avoid problems when trying to dry the biomass.biomass.Place the filter paper plus brine shrimp into a drying oven withPlace the filter paper plus brine shrimp into a drying oven with a temperature of a temperature of 36oC and allow it to dry for a minimum of 24 hours.36oC and allow it to dry for a minimum of 24 hours.After 24 hours, weigh the dried filter paper with biomass and reAfter 24 hours, weigh the dried filter paper with biomass and record this weight.cord this weight.Repeat sample preparation, enumeration, and biomass estimation fRepeat sample preparation, enumeration, and biomass estimation for each or each sample collected.sample collected.

Assessment of Cyst quantity in Lake Urmia during 2003Assessment of Cyst quantity in Lake Urmia during 2003

Average of Cyst distribution in different months (2003)

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35000

July Aug Sep Oct

Surface

WaterColumn