3. Materials and Methods
3.1 Study areas
Most of the chapters (I–IV) of this thesis have focused on lakes within the Värriö Natural Reserve, a natural reserve established in 1981. The Reserve covers an area of approximately 125 km2, between Salla and Savukoski municipalities, in the north-eastern side of Finnish Lapland bordering Russia. At the center of the reserve there is a small research station operated by the University of Helsinki. The station was built in the summer of 1967 and currently hosts several sub-arctic environment research projects. The use of motorized vehicles is strictly regulated in the park: it is allowed only for transferring heavy sampling gear and only on the route that connects the station to the nearest road. Other human activities, besides research, are strictly forbidden in the park.
Within the natural area there are several typical sub-arctic headwater lakes which are small (< 1 ha), with a simple bathymetry and vegetated, steep, shorelines. The catchment areas are also small (< 1.5 km2), as typical for headwater lakes, and covered by north-boreal coniferous forest dominated by Scots pine Pinus sylvestris (L.). The climate in the area is sub-continental, with an average annual mean temperature of -1 C° and an annual mean precipitation of about 600 mm. The lakes are dimictic, with an ice-cover typically lasting from mid-October to late-May.
Brown trout introductions in the reserve date back to 1980. Several lakes within the reserve were stocked at the same time, with adult brown trout from nearby populations (Pulliainen, personal communication), with the purpose of starting local recreational fisheries. Of all the stockings, brown trout introductions have been reported to be successful only in three lakes (Kuutsjärvi, Pirunkurulampi, Syväkurunlampi). However, only two of these fish populations (Kuutsjärvi and Pirunkurulampi) were found to be established at the time of the thesis work.
Different chapters of this thesis focused on two lakes:
• Lake Kuutsjärvi (67º 44’N, 29º 36’ E) I–IV) is in close proximity of Värriö research station and it is a small oligotrophic lake with a surface area of approximately 0.7 hectares. A small inlet enters the lake and a grid-blocked outlet exits it at opposite sides. The lake holds a self-reproducing population of brown trout.
• Lake Tippakurulampi (67º 46’N, 29º 37’ E) (III) is a fishless pond about 2 km north-east of the research station. It is small (about 0.2 ha), shallow and oligotrophic and is subject to extensive periods of depleted oxygen during the winter. Water in the lake has a slow turnover through a small
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mire area but the lake has no direct connection with other water bodies (water springs from, and drains to, the ground).
Lake Pirunkurulampi (67º45’N, 29º35’E) is oligotrophic (totP 4–7 μg l-1, totN 86–230 μg l-1), deeper and slightly dark colored (max depth 15 m and Secchi depth 7–7.5 m). It is located about 500 m distance from Lake Kuutsjärvi (I). Lake Pirunkurulampi had a very small brown trout population and it was possible to catch only few specimens for the studies, hence its marginal role.
Figure 1 - Lake Pirunkurulampi, a typical headwater gorge lake of northern Lapland – Picture © Kimmo Lahikainen
Water temperatures were recorded at two hours intervals from different depths with HOBO water temperature loggers throughout the study period (2010–2012). Temperature loggers were fixed on a rope attached to an anchored buoy; regularly spaced from the surface to the bottom at 2 m intervals (Kuutsjärvi) and 1 m intervals (Tippakurulampi) (IV).
Air temperatures were retrieved for the period 2010–2012 from the SMEAR station, located in the vicinity of the Värriö Research Station (II). Long-term air temperatures, precipitation and fallout of phosphorus (P) and nitrogen (N) were retrieved from the Sodankylä Meteorological station (about 130 km west from Lake Kuutsjärvi) (III, IV).
Table 1 – Limnological characteristics and average water chemistry parameters of Lake Kuutsjärvi, Lake Tippakurulampi and Lake Pirunkurulampi, from monthly measures throughout the study period (2010–2012), and list of samples collected for this thesis.
Parameter Lake Kuutsjärvi Lake Tippakurulampi Lake Pirunkurulampi
Area (ha) 0.67 0.22 ~ 0.6
Mean depth (m) 5.0 2.6 ~ 8
Maximum depth (m) 8.0 5.0 15
Secchi depth (m) 8.0 4.5 7
The last chapter (V) focused on a network of man-made and heavily managed canals in the Po river plain (Italy). The canals lie in a flat alluvial area, partly below the sea level, which is mainly used for agricultural purposes. In fact, the canal network spans over 4000 km of length in the Ferrara province alone and is mainly used to take water from the main river and bring it further inland to irrigate cultivations. The canals reach densities of 1.53 per km2 and undergo seasonal fluctuations in accordance to a balance between agricultural needs and the maintenance of minimum levels necessary for fish life. Heavy management practices, such as mowing banks and aquatic vegetation and the introduction of the grass carp Ctenopharyngodon idella (Valenciennes, 1844), has led to almost complete disappearance of the aquatic vegetation. The remaining vegetation is mainly represented by reed Phragmites australis (Cav.) and cattail Typha latifolia (L.) growing in a narrow strip along the banks. Besides grass carp, introduced voluntarily, a number of exotic species have colonized the area through natural dispersal from the Po river, only partially hindered by the existing pumps, siphons and weirs. Among these species were stone moroko Pseudorasbora parva (Temminck and Schlegel, 1846), pikeperch Sander lucioperca (L.), freshwater bream Abramis brama (L.), bitterling
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Rhodeus sericeus (Pallas, 1776) and, more importantly, wels catfish Silurus glanis (L.) and common carp Cyprinus carpio (L.). Exotic fish have thus interacted with native species such as Italian roach Rutilus pigus (Lacépède, 1804), Italian nase Chondrostoma soetta (Bonaparte, 1840), Italian barbel Barbus plebejus (Bonaparte, 1839), Italian golden loach Sabanejewia larvata (DeFilippi, 1859), three-spined stickleback Gasterosteus aculeatus (L.), chub Squalius cephalus (L.), Italian redeye roach Rutilus aula (Bonaparte, 1841), tench Tinca tinca (L.) and European perch Perca fluviatilis (L.), in a simplified habitat characterized by high human impact, occasional depleted oxygen and sharp water level fluctuations. 14 sites within this canal network, which were sampled over a long term (in 1991, 1997, 2003, and 2009) and for which environmental variables were recorded at regular intervals, were selected for this study. Hydrochemical data were obtained from the Regional Environmental Protection Agency of Emilia Romagna (ARPA), which monitors locations coinciding with, or representative of, water quality at each fish sampling site. Monitoring has been routinely performed since 1980 on a monthly basis, giving an ample coverage throughout the 18-years study period. The monitoring included temperature, specific conductance, pH, DO, BOD5, DIN, totP, totSS on all the samples.
Concentrations of Atrazine, Cd, Cr, Hg, Ni, Pb and Cu were measured frequently, but not uniformly (Table 2).
Figure 2- A typical artificial canal, running through an urbanized area and showing signs of partial naturalization – Picture © Giuseppe Castaldelli
Table 2 – Principal environmental parameters measured under long term monitoring of the canal network in the province of Ferrara, and presence-absence of native (N) and introduced (I) species sampled during the different years examined in the study. Values of environmental parameters are expressed as average, with corresponding intervals in parentheses.
Parameter Year
1991 1997 2003 2009
Temperature (ºC) 15.8 (2.1-30.4) 16.7 (2.7-31) 15.9 (0.5-29.8) 16.2 (1.1-30) Specific conductance (μs cm-1) 1308.5
D.O.(%) 75.5 (23-150) 75.5 (23-150) 81.1 (26-126) 82.3 (22-236)
BOD5 (mg l-1) 5.9 (1.5-18.3) 4.2 (1.5-16) 4.6 (2-18) 4.7 (2-26)
Anguilla anguilla (N) X X X X
Alosa fallax (N) X X
Rutilus pigus (N) X
Rutilus aula (N) X X
Squalius cephalus (N) X X
Tinca tinca (N) X X
Scardinius erytrophtalmus (N) X X X X
Alburnus alburnus (N) X X X X
Chondrostoma soetta (N) X X
Carassius auratus (I) X X X X
Rhodeus sericeus (I) X X
Pseudorasbora parva (I) X X X
Ctenopharyngodon idella (I) X X X X
Hypophtalmichthys molitrix (I) X
Sander lucioperca (I) X X X
D.O. – Dissolved Oxygen, BOD5 – Biochemical Oxygen Demand, DIN – Dissolved Inorganic Nitrogen, TP – Total Phosphorus, TSS – Total Suspended Solids
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3.2 Neolimnological dataFish data from small lakes
Fish samples were collected from Lake Kuutsjärvi during the open water periods of 2010–2012, in three sampling events at the beginning of June, July and August. Fishing time was varied trying to standardize the catch to about 10-15 specimens per catch event.
However, during August 2011, a mass biomanipulation effort involved the removal of the majority of the remaining fish population of Lake Kuutsjärvi. A few fish were left and caught in 2012, to test for density dependent effects (II).
Overall, the catches-per-unit-of-effort were maintained artificially low until August 2011 but declined sharply after the mass biomanipulation effort. During 2012 the CPUEs progressively declined even further and the brown trout population of Lake Kuutsjärvi was considered to be removed after one week without catches (IV).
Different gears were used in the sampling: multi-mesh gillnets, angling and a long-line.
Three types of gillnets were used: Fiskeriverket nets (30 m long and 2,4 m deep, with five 6 m panels of 17, 22, 25, 33 and 50 mm mesh size, knot-to-knot), Nordic nets (45 m long and 1.8 m deep, composed of nine 5 m panels of 10 12, 15, 19, 24, 30, 38, 47 and 60 mm mesh size, knot-to-knot) and a set of custom made nets (30 m long and 1.8 m deep, with five 6 m panels of 10, 15, 20, 25 and 30 mm mesh size, knot-to-knot).
Lake Pirunkurulampi was sampled with both gillnets and angling. Additionally, gillnets were deployed in Lake Tippakurulampi (and other putative fish-present lakes) to verify the presence/absence of fish. Angling was also used to tentatively assess potential fish presence in other remote lakes in the area.
All fish were frozen as quickly as possible (~ 15 minutes) at the field station and then transported frozen to the laboratory. Each fish total length (TL) was measured with a precision of 1 mm and weight with a precision of 0.1 g. Stomach contents were analyzed with a volumetric point method (Windell 1971). Each food item was identified as accurately as possible either to genus, family or sub-order level. Results of stomach content analysis (SCA) for subsets of the whole catch were used to standardize the number of samples from each month/year (I, II) whereas the whole dataset was used in later contributions (III, IV).
Fish age was estimated from otoliths and scales annuli (I, II, IV). Despite the high number of regenerated scales, it was always possible to find enough non-regenerated scales which could be used for ageing. However, often scales did not record the entire lifespan of the fish (only the first ~ 5 years), possibly because of the slow growth and the difficulties of identifying annuli too close to the edge of the scale. Whenever otolith and scale readings differed otoliths were considered more reliable and overruled scale readings.
Fish data from complex lotic ecosystems
Fish were also collected from 14 Italian canals, of comparable watershed, riparian and hydraulic characteristics, in 1991, 1997, 2003, and 2009 (V). All sampled stretches were confined between an upstream weir and a downstream pumping station; thus colonization was possible from upstream, but exit was effectively blocked.
Fish were sampled from mid-October to November, at the end of the irrigation season, when water depth reaches its annual minimum and most of the canal progressively empties, forcing fish to move to an area of suitable depth for fish life (0.6–1.3 m). Under these conditions it was possible to capture most of the fish community by using a seine net (Welcomme 1980) of 2 m in height with a 25 m mouth and a knot-to-knot mesh size of 8 mm. The cod-end was 3 m long and had a knot-to-knot mesh size of 4 mm.
A blocking net (4 mm knot-to-knot mesh size) spanning the canal width was used to impede fish escape and the seine was dragged towards it. After a first haul, a replicated haul was conducted on some occasions to check the recovery efficacy, which was always worse than 95% of species number and biomass of the first haul.
Fish were identified to species level, counted, measured (total length to nearest mm), and weighed (to nearest 0.1 g) before being released in another canal stretch. Abundances of fish species (individuals ha-1) were calculated assuming a surface area equal to the size of the canal stretch at mean water level. Biomass of fish species in catch (g ha-1) was calculated according to the same assumption.
Stable isotope and fatty acids data from small lakes
Samples for stable isotope analysis (SIA) were collected from liver and muscle tissues of each fish (I, II). Aquatic food web samples were collected during 2010–2012 using kick-nets, dip-nets, Ekman grab (all sieved through 500 μm mesh) and zooplankton nets (mesh sizes 50 μm and 150 μm). Sub-samples of aquatic invertebrates were kept for 24 hours in filtered spring water to flush their guts. Terrestrial samples were collected from the immediate surroundings of the lake using pitfall, light and screen traps, as well as butterfly nets for invertebrates and spring traps for vertebrates. A total of 13 taxa of putative prey from both the aquatic and terrestrial systems, spanning from invertebrates to vertebrates, were collected to quantify relative importance of aquatic and terrestrial energy sources. All samples were identified and stored frozen for further analysis. Each sample was freeze-dried (-49 ºC, about 0 Pa) and then ground to a fine powder in a mortar. About 0.25 mg of powder was then measured with a precision scale and used for measuring stable isotopic ratios of N and C using a Finnigan DeltaPlusAdvantage mass spectrometer (Thermo Scientific, Bremen, Germany) coupled to an elemental analyser NC 2500 (CE Instruments, Milan, Italy) via a ConFlow III interface (Thermo Scientific, Bremen, Germany).
For fatty acid analysis (FAA), a randomly selected sub-sample of fish (n = 15) and seven putative prey taxa (n = 15) captured in 2011 were used to evaluate the relative importance
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of aquatic and terrestrial sources of lipids in the brown trout diet (I). Approximately 5–10 mm3 of freeze-dried tissue were transmethylated to convert the acyl chains in the lipids of the tissue sample to fatty acid methyl esters (FAME). The samples were heated at 95 ºC in a methanolic 1% H2SO4 solution under nitrogen atmosphere for two hours, and the FAME formed were extracted with hexane in two steps. The dried and concentrated FAME of total lipids were identified and quantified by gas-liquid chromatography (GLC) (Käkelä et al. 2005). Altogether, peak areas of 55 different FAME were extracted from each chromatogram and converted to molar percentages. Subsequently, the fatty acids were treated as structural categories, which indicate prey origin and ecology well but are not prone to species-specific differences in fatty acid metabolism. Using such categories, two indices were calculated: the ratio of n-3 polyunsaturated fatty acids (n-3PUFAs) to n-6PUFAs (hereafter n-3/n-6 ratio) and the ratio of specific monounsaturated fatty acids (MUFAs) i.e. 16:1n-7 to 18:1n-9. The n-3PUFAs (a double bond in the third carbon calculated from the methyl end and the other methylene interrupted double bonds located further) dominate in the tissues of aquatic animals and n-6 PUFAs (a double bond in the sixth carbon from methyl end and the other methylene interrupted double bonds located further) in terrestrial animals. Despite the fact that the chain length and double bond contents of individual n-3 and n-6 PUFAs are modified by the metabolism of both prey and predator species, the n-3 and n-6 PUFAs cannot be interconverted. Thus, the tissue n-3/n-6 ratio of fish reflects the relative dietary supply of n-3PUFAs and n-6PUFAs remarkably well, as seen in brown trout feeding trials (Turchini et al. 2003). In the tissues of aquatic animals that live in cold temperatures, membrane-bound Δ9-desaturase inserts one double bond into de novo-synthesized or diet-derived 16:0, a saturated fatty acid, and thereby increases the fluidity of tissue lipids to meet environmental thermal demands.
In contrast, in terrestrial prey, living at higher temperatures during the sampling period (or throughout the year, e.g. homeothermic mammalians), the 16:0 first undergoes chain elongation to 18:0 and subsequently the double bond is added, which produces 18:1n-9 with a melting point higher than that of 16:1n-7. Therefore, selecting the fatty acid ratios n-3/n-6 and 16:1n-7/18:1n-9 filters taxonomic metabolic variability from the fatty acid data and allows better separation of aquatic and terrestrial energy sources.
Macro- and micro-invertebrate neolimnological data from small lakes
Macro- ( > 150 μm in length) and micro-invertebrates ( ≤ 150 μm in length) were sampled monthly (June–August) from both lakes using hoop plankton nets with a 30 cm diameter and mesh sizes of 150 and 50 μm (III). Three replicates per sampling event were taken at the deepest point of each lake, encompassing the whole water column. Qualitative samples were also collected from the pelagic area during night time to verify possible diel movements of the macro-invertebrates. Samples were immediately preserved with an 18 % formaldehyde solution and, later, the macro- and micro-invertebrate components of each sample were determined through a stereomicroscope. Samples were filtered of debris and excess water was dried, bulk wet weight was then measured with a precision of 0.01 g and density was expressed as a measure of weight per units of volume (mg l-1).
Qualitative samples were taken from the benthic and littoral domains to verify the presence of other macro-invertebrates in the lakes. Bottom samples (n = 5, per lake) were taken with a 25 x 25 cm Ekman bottom grab and sieved through a 150 μm mesh sized net, while littoral samples (n = 5, per lake) were taken with a handle-net (150 μm mesh size) swept over the submerged vegetation along the shoreline.
All limnological samples were collected in plastic screw cap containers with the addition of about 10 ml of an 18% formaldehyde solution. Samples were kept at low temperature 4-8 ºC after collection and until they could be examined in the lab.
3.3 Paleolimnological data from small lakes
Paleolimnological analyses were used to verify if theoretical and model-predicted effects of fish introductions could be confirmed in the sedimentary history of the lake.
Coring of sediments
Several surface sediment cores were retrieved through the ice in April 2009 using a HTH-Kajak type gravity corer (Renberg & Hansson 2008) (III, IV). Cores were taken from the deepest part of each lake, which were assumed to represent the highest accumulation areas. One core per lake was reserved for sediment dating, the other cores were sub-sampled for loss-on-ignition (LOI), macro- and micro-invertebrates, diatoms, and plant pigment analysis at intervals of 2.5 mm, representing a temporal resolution of ca. 1-10 years. A Limnos-type gravity corer was used to derive a 19.5 cm long sediment sequence from the deepest part of Lake Kuutsjärvi in spring 2011 and sub-sampled for LOI and stable isotopes of C and N at intervals of 5 mm. Sediment cores were sliced on the field in a dark room and each sub-sample was stored in labeled 0.25 l air-tight plastic bag.
Sediment sub-samples were stored at low temperatures (max 4 ºC) until further analysis.
Sediment dating and correlation
Freeze-dried sediment samples were analysed for 210Pb, 226Ra, and 137Cs by direct gamma assay in the Liverpool University Environmental Radioactivity. The CRS dating model (Appleby & Oldfield 1978) was then used to calculate the 210Pb chronology.
Correlation between the dating core and the other cores from the same lake was based on the analysis of organic content, through LOI (Heiri et al. 2001). A subsample of about 2 ml (0.5 g for already dried samples) of each layer was accurately weighted with a precision scale and then dried at 105 ºC for 16 hours. The subsample was then weighted again before and after a 550 ºC/4 hours ignition to evaluate the organic content.
Macro- and micro-invertebrate paleolimnological data
Sediments subsamples were treated with a 10% KOH solution for about 30 minutes, then rinsed and sieved trough a 40 μm filter. Subsequently they were mixed with a lycopodium
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tablet treated with a 10% solution of HCl and centrifuged at 3500 rpm for 4-5 minutes. The resulting sample was then mounted on a glass slide with safranine glycerol and analyzed trough a binocular microscope at 200X magnification. To account for dilution effects, influx rates (individuals cm-2 y-1) of macro- and micro-invertebrates were estimated using the known abundance and sedimentation rates (III). Macro-invertebrates subfossil remains such as head capsules (Chironomidae), mandibles (Gammarus lacustris (L.)) and ephippia (Daphnia longispina (L.)) were analysed as proxies of different ecological regions by treating samples with KOH and sieving ~ 3 ml sediment subsamples from Lake Kuutsjärvi. Macro-invertebrates influx rates were not available from Lake Tippakurulampi.
Micro-invertebrates (Cladocera) subfossil remains were analysed for both lakes from 1 mlsubsamples, using nomenclature from (Szeroczyńska & Sarmaja-Korjonen 2007).
Relative abundances of Cladocera species were estimated based on a minimum of 200 remains from each subsample, except for 9 subsamples from Lake Tippakurulampi that contained too low abundance of remains. The length of Eubosmina carapaces, mucri and antennulae were measured (Korosi et al. 2008) for every subsample from Lake Kuutsjärvi (n = 900). However, Eubosmina remains were too fragmentary in Lake Tippakurulampi, and hence it was possible to measure body sizes only for antennulae (n = 7–21) and mucri (n = 8–12) and for fewer subsamples (6 in total).
Diatoms
Diatoms were prepared using H2O2 digestion and HCl-treatment and cleaned diatoms were mounted in Naphrax® (Battarbee 1986) (IV). A minimum of 300 diatom valves from each sample were identified and counted along random transects at 1000x magnification.
Diatom identification was based mainly on Krammer and Lange-Bertalot (1986, 1988, 1991a).
Plant pigments
Pigments were quantitatively extracted from freeze-dried sediments in acetone: methanol:
water (80:15:5), filtered (0.2 μm PTFE), dried under nitrogen gas, re-dissolved into acetone, ion-pairing reagent and methanol (70:25:5) and injected into an Agilent 1200 series high performance liquid chromatography (HPLC) system (Leavitt & Hodgson
water (80:15:5), filtered (0.2 μm PTFE), dried under nitrogen gas, re-dissolved into acetone, ion-pairing reagent and methanol (70:25:5) and injected into an Agilent 1200 series high performance liquid chromatography (HPLC) system (Leavitt & Hodgson