ORGANOCHLORINE AND METAL ACCUMULATION IN FISH(PHOXINUS PHOXINUS) ALONG A NORTH-SOUTH TRANSECT IN

THE ALPS

RUDOLF HOFER1∗, REINHARD LACKNER1, JOACHIM KARGL1, BERTHATHALER2, DANILO TAIT 2, LEOPOLDO BONETTI2, RAFFAELE VISTOCCO2 and

GIOVANNA FLAIM 3

1 Institut für Zoologie und Limnologie, Universität Innsbruck, Austria;2 Landesagentur fürUmwelt- und Arbeitsschutz, Autonome Provinz Bozen;3 Istituto Agrario di San Michele (Trentino)

(∗ author for correspondence, e-mail: rudolf.hofer@uibk.ac.at)

(Received 19 January 1999; accepted 18 January 2000)

Abstract. Southern populations of the European minnow from remote oligotrophic mountain lakesalong an Alpine north-south transect accumulated more p,p′ DDE and PCBs than northern popu-lations. As these semi-volatile organochlorines predominantly evaporate in warm countries, higherrates of condensation (deposition) are assumed to occur in the southern slopes of the Alps. Thehigher accumulation of lead and cadmium in southern population is rather attributed to geogenic andspecific environmental impacts than to atmospheric deposition. Increasing liver ratios of [glutathionedisulfide]/[glutathione], a potential indicator for oxidative stress, from north to south reflect the gen-erally higher toxic load at remote sites at the southern edge of the Alps. However, histopathologicalchanges in the liver did not correlate with accumulated toxicants indicating that deposition does notlead to severe lesions but induces specific mechanisms for detoxification.

Keywords: histopathology, metals, oxidative stress, PCBs, p,p′ DDE

1. Introduction

Long-range atmospheric transport, precipitation and ‘cold-condensation’ of toxic-ants from human sources lead to a global distribution and contamination of en-vironments (Wania and Macky, 1993; Staciet al., 1995). Even in pristine ecosys-tems such as Antarctica surprisingly high concentrations of persistent chemicalshave accumulated. In addition, the low temperature in polar and mountain regionsslow down the biological degradation and excretion of toxicants in ectothermic or-ganisms. Extreme environmental conditions including low temperatures and shortvegetation periods combined with accumulated toxicants may lead to an additivestress to organisms inhabiting these environments (Psenner, 1989; Köcket al.,1995).

In this study we investigated the accumulation of selected organochlorines andmetals in the European minnow (Phoxinus phoxinus) from lakes along a north-south gradient in the Central Alps and their consequences for the health status offish.

Water, Air, and Soil Pollution125: 189–200, 2001.© 2001Kluwer Academic Publishers. Printed in the Netherlands.

190 R. HOFER ET AL.

TABLE I

Location and physical charcteristics of study lakes NT: North Tyrol; ST: South Tyrol; T: Trentino

◦North ◦East Altitude Lake Catchment Max. depth

(m) (ha) (ha) (m)

Zireiner See (NT) 47◦29′ 10◦50′ 1799 4.6 12 14

Blindsee (NT) 47◦28′ 11◦45′ 1092 29 574 24

Wildalpsee (NT) 47◦18′ 12◦09′ 1950 1.4 8 8

Langensee (NT) 47◦15′ 12◦00′ 2232 3.2 20 n.a.

Lichtsee (NT) 47◦02′ 11◦24′ 2104 0.8 11 8

Obernberger See (NT) 46◦59′ 11◦24′ 1590 12 1163 10

Pfitscherjoch See (ST) 46◦59′ 11◦39′ 2231 0.6 4 17

Schrüttensee (ST) 46◦45′ 11◦33′ 1957 2.2 99 n.a.

Durnholzer See (ST) 46◦44′ 11◦25′ 1560 12.4 2787 13

Pfaffensee (ST) 46◦43′ 10◦29′ 2222 1.7 39 6

Pragser Wildsee (ST) 46◦41′ 12◦05′ 1489 31 2664 36

Alplaner See (ST) 46◦27′ 10◦52′ 2387 3 50 20

Lago Malghette (T) 46◦19′ 10◦56′ 1891 9.5 413 11

Lago di Cavallazza (T) 46◦17′ 11◦47′ 2141 0.9 42 2

Lago di Tovel (T) 46◦16′ 11◦58′ 1115 737 3990 39

Lago Ritorto (T) 46◦13′ 11◦47′ 2056 7.7 26 25

Lago di Bombasel (T) 46◦13′ 11◦30′ 2267 1 45 2

n.a.: Not available.

2. Material and Methods

2.1. SAMPLING

During July and August 1997 adult European minnow (Phoxinus phoxinus) weretrapped in the littoral zone of 17 remote mountain lakes (1092–2387 m abovesea level) along an Alpine north-south transect from Northern Tyrol (Austria) toTrentino (Italy) (Table I). Two L samples of surface water were taken for routinelimnological analyses, and 100 mL filtered (0.45µm Millipore cellulose acetate)and acidified water (0.5 mL suprapure HNO3) for metal analyses. Lipophilic com-pounds in the water were not analysed as their concentrations were expected to beextremely low. Due to the remote status of the lakes sampling and concentrationof large volumes of water was not possible. About 80 fish from each lake werekilled immediately after capture and their body length was recorded. The head wasfixed in ethanol (age determination) and the intestine including the liver in 5%phosphate-buffered formalin (histology). The remaining body (carcasse) of eachfish was wrapped separately in aluminium foil and transported to the laboratory at

ORGANOCHLORINES AND METALS IN FISH FROM ALPINE LAKES 191

low temperatures where it was frozen. In addition, 20 live fish were transported inan aerated isolated container to the laboratory and dissected: The liver was frozenin liquid nitrogen and the head fixed in ethanol.

2.2. ANALYSES

Age determination:Opercular bones were soaked in diluted detergent for severalhours and cleaned of adherent tissue. The age of fish was estimated by countingthe number of yearly ridges formed during winter.

Analyses of organochlorines (Bernie and Grimalt, 1998): Three pools of 6–8fish carcasses of the same age (5, 6 and 7 yr) were freeze dried and extractedwith n-hexane dichloromethane (4:1) for 18 hr. After evaporation of an aliquot ofthis extract (10%) the lipid content of the sample was weighted. The rest of theextract was spiked with TBB and PCB209 (for assessing the analytical recovery),the volume reduced to 2 mL and mixed with sulphuric acid for cleaning the extract.After stirring, the two layers were separated by centrifugation and the sulphuricacid removed. The n-hexane extract was neutralised by washing three times withMilli-Q water and concentrated under vacuum. Samples (2µL) were analysed in agas chromatograph equipped with a63Ni electron capture detector, a DB-5 column(5% phenyl, 95% methylpolysiloxane) and a split/splitless injector. Helium wasused as the carrier gas (30 cm sec−1). Two µL of samples were introduced withan automatic injector. Injection and detector temperatures were 270 and 310◦C,respectively. Oven temperature was programmed from 60 to 300◦C at 6◦C min−1

with a final holding time of 10 min. The make up gas was nitrogen (60 cm sec−1).Metal analyses:5–10 pools with 4–5 fish carcasses of the same age (4–8 yr)

were dried to constant weight at 40◦C and homogenized. An aliquot of 0.5 g wasdigested by addition of 6 mL suprapure HNO3 (65%) and 1 mL H2O2 (30%) in amicrowave oven. Metal analyses (Hg, Se, As, Cd, Pb, Co, Ni, Cu, Zn, Mo, Mn)were performed by graphite furnace atomic absorption spectrophotometry. Lobsterhepatopancreas, mussel, dogfish liver and dogfish muscle were used as certifiedstandard tissues.

Biochemical analyses:Determination of glutathione (GSH) and glutathione di-sulfide (GSSG). Small pieces of liver (5–20 mg) were dissected from the frozenintestines and homogenized in 200µL ice-cold 10% metaphosphoric acid (w/v).Homogenates were centrifuged (10 min at 13 000 r.p.m) and the supernatants usedfor the determination of GSH and GSSG. The protein content of the pellet wasdetermined by the method of Lowryet al. (1951).

GSH and GSSG from the fish samples were separated on a Merck LiChrospherRP select B column, 0.4× 25 cm, by using 0.1% TFA (trifluoro acetic acid) aseluent at 1 mL min−1. Detection was carried out by post column reaction using 2solutions added sequentially at 0.5 mL min−1: (1) 0.55 M NaOH + 5% diethano-lamine were added to the eluent. (2) After passing the mixture through a reactioncoil at 100◦C, 200 mg L−1 OPA (o-phtaldialdehde, dissolved in 4 mL dimethyl-

192 R. HOFER ET AL.

Figure 1. Separation of GSH and GSSG in extracts of fish liver (A) and a standard containing0.05 mM L−1 GSH and GSSG (B). Injection volume was 10µL, detection was carried out flu-orimetrically at 340/420 nm. Peaks appearing in the chromatogram in some samples prior to GSHmay be attributed to sulphur containing amino acids.

formamide) in 0.5 M sodium phosphate buffer, pH 7.0, were added as fluorigenicreagent. Detection was carried out fluorimetrically at 340/420 nm after passingthe mixture through another reaction coil at room temperature. This new methodallows the simultaneous determination of GSH and GSSG without the need toderivatize the samples prior to analysis (Figure 1). GSH and GSSG concentrationsin the metaphosphoric acid samples where found to be stable for at least 6 hr whenkept cold.

Histology: Pieces of liver were gradually dehydrated in ethanol and embed-ded in methyl methacrylate. Microtome sections (3µm) were stained with May-Grünwald/Giemsa and evaluated by microscopy. Selected samples were subjectedto PAS (glycogen) and Sudan black (lipid) staining. For the latter formalin fixedsamples were transferred to 30% sucrose, coated with ‘tissue tek’, shock frozenand cut with a ‘kryocut’. The method of Weibel (1979) was applied for quantitativeevaluations of pathological changes.

Water analyses:pH, conductivity, alkalinity, ammonia, nitrate, sulphate andphosphate were determined following routine methods. Ions were analysed witha Dionex-120 ion chromatograph. Metal analyses were performed by InductionCoupled Plasma Mass Spectrometry. For quality assurance five different certifiedstandard water samples were used.

OR

GA

NO

CH

LOR

INE

SA

ND

ME

TALS

INF

ISH

FR

OM

ALP

INE

LAK

ES

193

TABLE II

Selected chemical characteristics of study lakes

pH Cond. Alk. Ca2+ P (ges.) NO−3 Pb Cd Ni Cu Zn Al

µS cm−1 meq L−1 mg L−1 µg L−1 µgN L−1 µg L−1 µg L−1 µg L−1 µg L−1 µg L−1 µg L−1

Ziereiner See 8.14 171.6 2.02 32.37 3.5 0.066 0.02 0.003 1.10 0.05 7.2 6

Blindsee 8.22 268.2 3.04 35.01 3.2 0.099 0.02 0.003 1.50 0.05 3.3 3

Wildalpsee 7.59 21.0 0.15 3.83 6.8 0.035 0.11 0.010 0.18 0.05 3.3 15

Langensee 6.83 11.0 0.10 1.30 6.8 0 1.80 0.080 0.73 2.60 90.3 26

Lichtsee 7.46 25.1 0.21 5.02 8.2 0 0.06 0.005 0.09 0.05 1.0 15

Obernberger S. 8.20 162.7 1.74 26.23 4.1 0.247 0.04 0.010 1.90 0.05 48.3 3

Pfitscherjoch. S. 7.33 11.2 0.08 1.40 6.0 0 0.11 0.010 0.12 0.19 78.7 6

Schrüttensee 7.56 48.5 0.33 8.50 2.0 0.127 5.30 0.050 0.85 1.90 286.0 32

Durnholzer S. 6.80 34.5 0.10 4.10 3.0 0.227 1.40 0.060 1.30 1.80 52.5 15

Pfaffensee 7.15 36.6 0.14 2.70 12.0 0.025 3.40 0.100 2.10 19.40 316.0 63

Pragser Wildsee 8.42 202.0 2.31 28.90 2.0 0.379 1.00 0.050 2.10 1.10 20.7 11

Alplaner See 7.47 31.1 0.20 4.48 4.0 0 3.30 0.040 0.69 5.90 88.4 4

L. Malghette 6.30 13.8 0.05 1.50 9.0 0.143 0.15 0.020 0.23 0.40 2.3 16

L.d. Cavallazza 6.40 16.1 0.11 2.30 10.0 0.008 0.41 0.030 0.33 0.69 121.0 32

L.d. Tovel 7.90 178.0 2.02 28.50 10.0 0.287 0.22 0.040 1.00 0.96 2.7 1

L. Ritorto 6.50 20.0 0.12 2.30 6.0 0.210 0.70 0.070 0.19 7.70 10.7 11

L.d. Bombasel 6.50 11.6 0.06 1.10 6.0 0.418 0.18 0.030 0.17 0.14 2.2 1

194 R. HOFER ET AL.

Figure 2.p,p′ DDE concentration of fish carcasses (Phoxinus phoxinus) along the Alpine north-southtransect. Open symbols are for fish from hard water lakes, closed symbols for softwater lakes.

3. Results

3.1. CHEMICAL CHARACTERISTICS OF STUDY LAKES(TABLES I AND II)

All lakes, 5 hardwater lakes (>26 mg L−1 Ca2+) and 12 softwater lakes (<9 mgL−1 Ca2+), are oligotrophic with low concentrations of nutrients and monovalentions (Na+ <1 mg L−1, Cl− < 0.5 mg L−1). Softwater lakes of Trentino had pHvalues slightly lower than 7, reflecting the geology of their catchment; all otherlakes were neutral to alkaline. Aluminium concentrations were low or only slightlyelevated. Concentrations of trace metals were generally low and correlated roughlyamong each other but not with water hardness and pH. The highest values werefound in Schrüttensee (Pb, Zn) and Pfaffensee (Cu, Zn, Al). All lakes are locatedat remote sites, i.e. there is no direct anthropogenic import in the catchment.

3.2. TOXICANTS ACCUMULATED IN FISH

p,p′ DDE, a major metabolic product of DDT accumulating in the lipid fraction offish carcasses, correlated significantly with the geographical latitude of the lakes,with one order of magnitude higher values at the southern border of the Alps (Fig-ure 2). Lipid contents of samples varied but did not show a north-south gradient.

ORGANOCHLORINES AND METALS IN FISH FROM ALPINE LAKES 195

Figure 3.Correlation between the concentrations of p,p′ DDE and total PCBs in individual pools offish carcasses.

The accumulation pattern of total PCB’s correlated with the p,p′ DDE content offish (Figure 3). However, as sample volumes were relatively small, PCB valuesof some lakes were below detection limits. Organochlorine concentrations in fishwere found to be independent of the altitude of the lake and did not correlate withany of the water parameters.

The concentration of trace metals in the water did not correlate with metalconcentrations in fish. Minnow from soft water lakes accumulated more Pb and Cdthan those from hard water lakes (Figure 4). Exceptional high concentrations of Cdand Pb were found in fish from Pfitscherjochsee. The minnow of other softwaterlakes but not those of hardwater lakes indicate a north-south gradient which isless pronounced than that of p,p′ DDE. Other trace metals are scattered along thetransect and do not show significant differences between hard and soft water lakes.A significant age-dependant accumulation of trace metals in fish carcasses wasfound only for populations with high concentrations of mercury which increasedby 100% (0.5–1.0µg Hg g−1 dry weight) from four to eight years old fish.

196 R. HOFER ET AL.

Figure 4.Cadmium (A) and lead (B) concentrations of fish carcasses along the north-south transect.Open symbols are for fish from hard water lakes, closed symbols for softwater lakes. Note the excep-tionally high concentrations in Pfitscherjochsee. Only fish from softwater lakes show an increasingtrend of metal accumulation towards the south.

ORGANOCHLORINES AND METALS IN FISH FROM ALPINE LAKES 197

Figure 5.Oxidative stress as indicated by the glutatathione disulphide (GSSG) vs. glutathione (GSH)ratio of minnow liver along the north-south transect. Open symbols are for fish from hard water lakes,closed symbols for softwater lakes.

3.3. BIOCHEMISTRY AND HISTOLOGY OF THE LIVER

The concentration of both gluthathione disulfide (GSSG) and the GSSG/glutathioneratio in the liver of minnow significantly increased from north to south (Figure 5),comparable to the pattern of p,p′ DDE.

The most striking pathological change in the liver of some minnow popula-tions were focal hydropic degenerations (vacuolization) of hepatocytes which oc-curred as single cells or as cell aggregates. Although the populations from Lagodi Bombasel and Lago di Cavalazza, two lakes at the southern end of the transekt,displayed by far the highest degree of hydropic degeneration, no significant cor-relation with the geographic latitude was found. However, the minnow from thesetwo lakes accumulated high amounts of Nickel (Figure 6).

Other pathological changes such as focal inflammations, necroses of single hep-atocytes or proliferation of bile ducts were found occasionally or were only mildand did not correlate with accumulated toxicants or with GSSG.

198 R. HOFER ET AL.

Figure 6.Correlation between nickel concentrations in fish carcasses and the fractional area of hy-dropic degenerated hepatocytes in the liver of minnow. Open symbols are for fish from hard waterlakes, closed symbols for softwater lakes.

4. Discussion

The atmospheric transport of organic compounds largely depends on their va-por pressure (Wania and Mackay, 1993). Involatile substances such as benzo-[a]-pyrene usually remain in the region of its emission while highly volatile com-pounds, e.g. hexachlorobenzene, are transported over long distances. Semi-volatileorganochlorines, e.g. DDTs and higher chlorinated PCBs, however, preferably con-dense in temperate regions with decreasing accumulation towards the poles. As aconsequence, the volatility of compounds and the ambient temperature stronglyinfluence the distribution pattern of organic toxicants. Recently it has been shownthat this characteristic temperature-dependent distribution pattern of organic com-pounds is also valid at a local altitudinal scale (Blaiset al., 1998). The snowat high altitudes contained the highest portion of low molecular (volatile) PCBswhereas high molecular PCBs are more abundant at lower altitudes. Peripheralregions of mountains receive more precipitation than central parts and decreasingtemperatures in ascending air favour the condensation of low volatile compounds(Barros and Lettenmaier, 1993). As a consequence, exposed slopes receive moretoxicants than central parts or protected areas with less precipitation. Fish are long-living animals accumulating toxicants integrating over time and space. p,p′ DDEconcentrations in the liver of burbut (Lota lota) caught along a Canadian north-

ORGANOCHLORINES AND METALS IN FISH FROM ALPINE LAKES 199

south transect from 67 to 50◦ increased by a factor of 2.5 (Muiret al., 1990).Minnow from the southern parts of the Alps contain even 10 times more p,p′ DDEthan those from the northern edge, although the distance is relatively short (1.2◦;130 km). The low contamination of northern populations can be explained by thepredominant origin of p,p′ DDE. Due to the more frequent application and higherambient temperatures DDTs predominantly evaporate in southern countries andcondense along the slopes of southern Alps. Circulation from north-west, however,may be expected to contain lower concentrations of semi-volatile organochlorines.In contrast to snow analyses of Blaiset al. (1998), our results with fish did not showany altitudinal dependence of p,p′ DDE accumulation. This is probably due to therelatively small range of altitudes (1000–2300 m above sea level) selected along theAlpine transect. Although DDT has been banned in European countries since the1970s, its degradation products are still present in all ecosystems (Wiktelius andEdwards, 1997). Some developing countries, however, still use DDT. Although de-gradation products of DDT, in particular p,p′ DDE, have lost their insecticide effect,they impair, together with other organochlorines, the reproduction of many animals(Blus, 1995). Contaminated birds may suffer a complete loss of reproduction dueto the reduced thickness of egg shells.

p,p′ DDE and PCB deposition at the southern border of the Alps indicates thatalso other semi-volatile organic compounds may be present at elevated levels. Inaddition, fish from the southern Alps accumulated more lead and cadmium thanthose from northern parts. Although atmospheric deposition of metals is well doc-umented (Nriagu, 1990) their geogenic origin might be of higher importance. Inparticular, the high concentrations of lead and cadmium in minnow from Pfitscher-jochsee seem to be associated with ancient mining activities in this area. Further-more, several environmental factors strongly influence metal accumulation. Softwater, higher temperatures and in some cases also acidification favour metal uptakeby fish gills (Köck and Hofer, 1998). In fact, southern parts of the Alps have awarmer climate, and the lakes with the lowest water pH were situated in the south.

The GSSG/GSH ratio of the liver, though being unspecific, is a more significantindicator for the higher toxic load of southern fish populations than the accumu-lation of selected toxicants (Lackner, 1998). The toxic action of many substancesis linked with oxidative stress, i.e. the formation of reactive oxygen compoundsdamaging DNA, proteins and lipids (Kappus, 1987). To avoid biochemical andmorphological lesions several mechanisms have been evolved to detoxify theseradicals (Lackner, 1998). Glutathione is one of the most potential radical scav-engers being oxidized to GSSG. Thus, the accumulation of GSSG in tissues is asensitive indicator for oxidative stress. Although southern populations of minnoware contaminated, the toxic load is apparently within tolerable limits since patho-logical changes of the liver did neither correlate with the accumulation of toxicantsnor with the GSSG content of the liver. The hydropic degeneration of hepatocytes,the only severe liver lesion seen in minnow from the lakes investigated, correlatedonly with the nickel content of the fish.

200 R. HOFER ET AL.

5. Conclusion

Environments of even remote sites of the Central Alps may be highly affected byatmospherically deposited semi-volatile organochlorides. Especially the southernslopes seem to accumulate significantly more toxicants than the northern ones, asreflected by higher concentrations of p,p′ DDE and symptoms of oxidative stressin minnow populations of mountain lakes.

Acknowledgements

This research was supported by the ‘Österr. Ministerium für Wissenschaft undVerkehr’, the ‘Landesagentur für Umwelt- und Arbeitsschutz (Autonome Prov-inz Bozen)’ and the ‘Istituto Agrario die San Michele (Trentino)’. We gratefullyacknowledge the technical assistence of G. Sonntag, J. Franzoi and V. Pinamonti,and the fishing permits. We thank, U. Nickus, R. Tessadri, T. Braunbeck and R.Dallinger for discussing our results and R. Psenner for critical reading the manu-script.

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