MEASURING AND MODELLING THE DYNAMIC RESPONSE

OF REMOTE MOUNTAIN LAKE ECOSYSTEMS TO

ENVIRONMENTAL CHANGE

A programme of MOuntain LAke Research

Norwegian Institute for Water Research, Oslo

Universite de Bordeaux I et CNRS, Arcachon

University of Innsbruck

Department of Environmental Chemistry (CID-CSIC)

Protocol for fish population sampling

Bjrrn Olav Rosseland, NIVA, Norway

In AL:PE 1 and 2, the gillnet series that was design for the Norwegian monitoring programme (Rosseland et al. 1979) has been used (Wathne et al. 1995). Eight individual bottom gillnets of different mesh sizes (Table 1) or a set of three gillnets, each as a combination of these 8 mesh sizes (SFT 1983), has been used either alone or in combination.

The new series consist of three gillnets, 32 X 1.5 m, each net containing the 8 mesh sizes mixed and randomised in units of 4m. The same type of nets as in the single net SNSF series (thread size, colour etc.) are used, as well as the producer of the nets. Because of the different catch efficiency of the series, the old (8 single nets) was given a Catchability of 1, compared to 0.46 of the multimesh size series (3 nets).

Mesh size mm. 10 12.5 16.5 22 25 30 38 46

Thread thickness mm. .15 .15 .15 .15 .15 .15 .15 .17

Multimesh-size gillnet series has also been used in other Nordic countries. In Sweden, a monitoring program was initiated in 1983 to study the long-term effects of liming on fish populations (Degerman et al. 1988). In this program, a multimesh-size gillnet series containing 14 randomly distributed panels of 4 x 1.5 m (total gillnet length 42 m) of mesh sizes from 6.25 to 75 mm was used (Hammar and Filipsson 1985).

In 1990, a co-operation between Norway, Sweden and Finland was established to develop sampling protocols for monitoring of fish populations in acidified lakes. As a part of this, a new "Nordic multimesh-size gillnet series" was developed, which since then has been tested against the "national standard" for each country. In Finland, the comparative studies started in 1993 (Kurkilathi and Rask, 1995), and in Norway in 1992 (Jensen and Hesthagen 1996, SFT 1993). The Nordic series contains

12 different mesh sizes, between 5 and 55 mm,, in panels of 2.5 m, height 1.5 m and total length of 30 m per net, Table 2.

Mesh size mm. 5 6.25 8 10 12.5 16 19.5 24 29 35 43 55

Thread thickness mm. 0.1 0.1 0.1 0.12 0.12 0.15 0.15 0.15 0.15 0.18 0.20 0.25

In 1992 (SFT 1993, Figure 2), a comparative study on 8 brown trout and 5 Arctic charr (Salvelinus alpinus L.) populations in Norway, found the comparative CPUE values between the old SNSF series and the Nordic series to be:

In another comparative study using the SNSF and the Nordic series, the conclusion was stated:, "Over a limited range of mesh sizes and fish sizes (5-20 cm fish), the selectivity of the two types of net do not differ much" (Jensen and Hesthagen 1996).

Based on these results, there seems to be no problem by either the SNSF series or the Nordic series as standard gillnets in the MOLAR.

The gillnets are set perpendicular to the shore, avoiding steep-slope shore to bottom areas. Gillnets in the "old" series are set at random as for mesh size, whereas the "new" series always will have all eight mesh sizes represented at a certain point.

Both in the AL:PE 1 and 2 projects, the period between August 15 to October 15 have been selected for testfishing, for reasons given in Wathne et al. (1995). This is the main (and only) period for providing data to describe the population structure of salmonids. However, in the MOLAR project, physiological and histological data from other periods of the ice free season are needed. Sub-sampling of fish will therefore take place in some localities from early summer to late autumn.

Each fish must be given a specific number which follow all subsamples to be analyzed. Data from individual fish must be sent to NIVA. Due to differences in age determination of fish from some lakes in the AL:PE 2 project, age determination of all fish used in the MOLAR project should be undertaken by one laboratory.

For each individual fish, the following parametres must be noted:

· lake

· date

· species

· length, in mm, measured from snout to lower part of tail.

· weight, in gram.

· sex and gonadal maturation, from stage I - VII

· flesh colour; white, pink or red.

· stomach fullness, classified from 0 - 4.

· stomach content (if possible), conserved in 70% alcohol and classified in main invertebrate groups (not fully analysed in AL:PE 2).

· scale samples from brown trout only for age determination, taken from the area between the sideline organ and dorsal and pectorial fin.

· otolith samples for age determination (all species), using the "burning technique" described by Christensen (1964). If age differ when determined by otolith and scales, otolith age is considered as the true age.

· growth, determined by:

- length at catch as a function of true age.

- back calculation of growth (brown trout only), using the methods of Dahl (1910) and Lee (1920).

Bjrrn Olav Rosseland

NIVA

Brekkevein 19

P.O. Box 173 kjelsls

N-0411 Oslo, Norway

Phone: 47 22185110

Fax: 47 22185200

bjoern.rosseland@niva.no

Protocol for fish physiology

Jean-Charles Massabuau, CNRS, Arcachon, France

To avoid intercalibrations, all blood ion analyses will be performed at CNRS, Arcachon, France. CNRS will send a complete package to all MOLAR Fish Groups containing sampling and storing equipment.

Each kit contains either 1.25 or 2.5 ml of Drabkin solution. It is toxic as it contains cyanide. So take care although the risk is really minimum with these kits. You can use the provided gloves. See procedure on the video and on the back side of the general cooking list.

Contact us before sending

Jean-Charles MASSABUAU Tel: (33) 56 22 39 25/ (33) 56 22 39 20

Laboratoire de Neurobiologie et Physiologie Comparees Fax: (33) 56 83 03 50

Universite Bordeaux I et CNRS e-mail: massabuau@lnpc.u-bordeaux.fr

Place du Dr Peyneau

33120 Arcachon France

Jean FORGUE Tel: (33) 56 83 03 28

Laboratoire de Neurobiologie et Physiologie Comparees Fax: (33) 56 83 03 50

Universite Bordeaux I et CNRS e-mail: forgue@lnpc.u-bordeaux.fr

Place du Dr Peyneau, 33120 Arcachon France

- tweezers and scissors You must have your own material

On the field, prepare solutions 1 and 2 below, USE LATEX GLOVES:

Cacodylate buffer: Solution 1 in bottle 1,

- Reciepe. To prepare a few hours before the test fishing.

You have a plastic bottle with 97.5 ml of distilled water in it. There are 1.07g of sodium cacodylate in the small plastic can that is scotched on it. Empty it in the distilled water and put also the can (and its plug) in the water. Sheake it to dissolve the powder. Add the 2.5 ml of HCl 0.2 N provided in the 2 ml syringe. Sheake it. The volume of acid has been calculated to adjust the pH of the solution at 7.4.

Solution 2 in bottle 2: break one 10 ml ampula of glutaraldehyde and mix with 10 ml of cacodylate buffer by using 10 ml syringes,

Solution 3 in bottle 3: break one 2 ml ampula of osmium tetroxide and mix with 6 ml of distilled water by using 10 ml syringes.

Jean-Charles MASSABUAU Tel: (33) 56 22 39 25 / (33) 56 22 39 20; Fax: (33) 56 83 03 50

e-mail: massabuau@lnpc.u-bordeaux.fr

Suzanne Dunel / Claudine Chevallier Tel: (33) 88 10 69 00; Fax: (33) 88 10 69 06

Protocol for fish histology

Rudolf Hofer, Institute of Zoology, UIBK, Austria

2. Dissection of the fish

Dissect the fish immediately at the lake site.

You will get a complete set containing:

a. Tubes with formalin for tissues (all tissue samples of one fish are collected in one tube)

b. Empty tubes for otoliths

c. Figures for fish dissection

d. Protocol form

Protocol for heavy metals in fish

Sigurd Rognerud, NIVA, Norway

To get an estimate of adjusted means with an acceptable statistical significance it is necessary to collect 25 fish of different length in the intreval 15-30 cm from each lake. Record total length, weight and sex. Collect scales or otoliths for age estimates. Remove approximately 40g of bone and skinnless dorsal axial muscle, liver and kidneys from each fish, put it separatly in polyethylene bags and store it in the freezer.

All samples must be carefully packed and shipped to NIVA in a frozen condition. Inform the reciever so the package could be picked up before it thaws.

Approximately 1g muscle is digested in a 4: 1 nitric-perchloric acid mixture under pressure. After dissolution, samples are analyzed for mercury by a atomic absorption spectrophotometer equipped with a hydrid generator (detailed description given in Fjeld & Rognerud 1993). Lead and cadmium are analysed by atomic absorption spectrometry using a graphite furnace.

Sigurd Rognerud¹⁾ or Leif Lien²⁾

NIVA

P.O. Box 173 Kjelsls

N-0411 Oslo, Norway

Phone: ¹⁾ 47-62576400 ²⁾ 47-22185100

Fax: ¹⁾ 47-62576653 ²⁾ 47-22185200

sigurd.rognerud@niva.no

leif.lien@niva.no

Protocol for organic micropollutants in fish

Joan O. Grimalt, Lourdes Berdie, Barcelona, Catalonia, Spain

1.1 Compounds to be determined.

* Hexachlorobenzene

* Hexachlorocyclohexanes (namely a and g isomers)

* DDTs (namely, pp'-DDE and pp'-DDT)

* Polycyclic aromatic hydrocarbons (PAH; 63 individual compounds)

* Water and lipid content

-Self adhesive labels

-Pen

-Big box to keep the samples when dissected

-Field fridge

-Glass bottles

-Recipient for cleaning and rinsing residues

-Plastic tray.

-Scalpel

-Tweezers

-Scissors

-Aluminum foil

-Sulphuric acid for analysis grade (Merck)

-Potassium hydroxide (for analysis, Merck)

-Iso-octane (for trace organic analysis, Merck)

-Acetone (for trace organic analysis, Merck)

-Distilled water

-Lake water

-Soap (Extran MA01, ref. 7555.1000, alkaline, Merck)

-Internal standards: Tetrabromobenzene and PCB 209

Correct cleaning of the material is a very important step in the analysis of organic compounds. Plasticizers are present in most recipients and material of common use (e.g., gloves, bottles -distilled water is usually kept in plastic bottles-, ...). The hands and hair can also be a source of organic compounds in the sample. For this reason the cleaning procedure needs to be followed carefully during sampling and analysis.

1. Find a flat horizontal surface to work comfortably.

2. Put the gloves on.

3. Fill the plastic tray with EXTRAN solution (20 g per litre of distilled water).

4. Clean the glass bottle with this solution.

5. Rinse the glass bottle with lake water (3 times)

6. Fill the glass bottle with lake water.

7. Spread an aluminium sheet over the grass.

8. Put in the tray the material to be used and clean it.

9. Once cleaned, rinse it with the lake water stored in (6).

10. Put the material on the aluminium sheet spread in (7)

11. Cover the clean material with aluminium if not to be used immediately.

5 g. of muscle. 0.5 g of liver.

The fish tissues, protocol form and the list of analyses performed to each fish should be placed in a box containing dry ice and sent to Barcelona. Before mailing contact directly with Joan Grimalt, Pilar Fernandez or Rosa Vilanova:

Department of Environmental Chemistry (CID-CSIC)

Jordi Girona, 18

08034-Barcelona

Catalonia. Spain

Phone: 34 3 400 61 22 or 34 3 400 61 00

Fax 34 3 204 59 04

e-mail: jgoqam@cid.csic.es

lbrqam@cid.csic.es

mrvqam@cid.csic.es

pfrqam@cid.csic.es

1.8 Analyses (extraction).

The tissues are freeze-dried in an oil-free freeze-drier and Soxhlet extracted with (4:1) n-hexane-dichloromethane for 18 hours. An aliquot (10%) of this extract is used to measure total extractable lipid weight after evaporation to dryness. Water content is determined by weight difference prior and after freeze-drying.

The extract is spiked with TBB and PCB209 in order to assess the analytical recovery. Then it is reduced to 2 ml of n-hexane and cleaned up with agitation with sulphuric acid. After vigourous stirring in a Vortex (2 min) the two layers are decanted by centrifugation for removal of the sulphuric acid. The n-hexane extract is neutralized by washing (three times) with Milli-Q water for pH neutralization. The n-hexane is then concentrated under vacuum to a small volume, e.g. 50 l, for instrumental analysis.

After lyophilisation, the sample is saponified with 6 N NaOH and then extracted with (4:1) n-hexane-dichloromethane to obtain the total organic extract. The extract is vacuum evaporated to 0.5 ml and fractionated by column chromatography using columns filled with 5% water deactivated silica (top) and alumina (bottom). The first fraction (n-hexane) is discarded. The second fraction (n-hexane:dichloromethane) is collected and evaporated (vacuum rotary evaporation and nitrogen stream) to dryness for PAH analysis.

A gas chromatograph Hewlett-Packard Model 5890 (Palo Alto, CA, USA) equipped with a ⁶³Ni electron capture detector and a split/splitless injector is used. This equipment is provided with a DB-5 column (5% phenyl-95% methylpolysiloxane, 25 m length, 0.25 mm i.d., 0.25 mm film thickness; J&W Scientific, Folsom, CA, USA). Helium is the carrier gas (30 cm/s). The samples (2 l) are introduced with an automatic injector Hewlett-Packard Model 7673A in the splitless mode. Injection and detector temperatures are 270 and 310sC, respectively. Oven temperature is programmed from 60 to 300sC at 6sC/min with a final holding time of 10 min. The make up gas is nitrogen (60 cm/s).

A Carlo Erba GC8000 Series coupled to a mass spectrometer Fisons MD800 is used. This instrument is equiped with a 30 m HP-5 column coated with 5% phenyl methyl silicone. The oven temperature program is from 90 to 310sC at 4sC/min, held for 10 min. Injection and transfer line temperatures are 280 and 300sC, respectively. Helium is the carrier gas (50 cm/s). Data are acquired in the electron impact mode with an electron energy of 70 eV and the operation is in selected ion monitoring. The injection is in the splitless mode (1 l injected; hot needle technique), the split valve being closed for 48 s.

1.13 Quantitation.

Authentic standards of hexachlorobenzene, a-, g- and d-hexachlorocyclohexane, op'-DDE, pp'DDE, op'-DDD, pp'-DDD, op'-DDT, pp'-DDT and the polychlorobiphenyl congeners Nos. 28, 52, 101, 118, 138, 153 and 180 are used. Calibration curves (detector response vs amount injected) are performed for each compound. The polycyclic aromatic hydrocarbons are analyzed by reference to standards of fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[k]fluoranthene, pyrene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, benzo[ghi]perylene and dibenzo[ah]anthracene. The range of linearity of the detector is evaluated from the curves generated by representation of detector signal/amount injected vs amount injected. All measurements are performed in the ranges of linearity found for each compound. In some cases, re-dilution and re-injection are performed to fit within the linear requirements.

1.14 Compound identification.

Structural identification is confirmed by analysis of selected samples by gas chromatography coupled to mass spectrometry in the chemical ionization mode and record of the negative ions. The chromatographic conditions are the same as described above. A DB-5 column was used. Transfer line and ion source temperatures are 300 and 120sC, respectively. The reagent gas is methane. Data are acquired by scanning from 50 to 500 mass units at 1 s per decade. The samples selected for GC-MS analysis must allow to elucidate the composition of all the major peaks present in the gas chromatograms obtained with the ECD.

Samplig protocol forms for lakes Gossenkollesse, Redo and Rvre Neadalsvatn.

Samplig protocol forms for the lakes where only 5 fish specimens will be taken.

List of analyses available for each fish

List of analyses available for each fish