perennially frozen 
 and glacierized high - alpine areas react very 
 sensitively to changes in air temperature and even small differences 
 in temperature determine their frozen status . as a consequence ,
glaciers 
 in the european alps , for example , have lost 3040% of surface 
 area since the end of the little ice age ( middle of 19th century ) , and the lower permafrost limit is estimated to 
 have risen by 1 m per year .
discharge 
 from melting glaciers and thawing permafrost can be highly enriched 
 in solutes due to prolonged interaction with fine - grained rocks and 
 freshly exposed mineral surfaces .
solute outflows from 
 catchments characterized by cryospheric features can cause serious 
 changes in water chemistry downstream .
a survey of remote high altitude 
 lakes in the european alps has provided 
 strong evidence of a substantial increase in base cation and sulfate 
 concentrations in lake water over the past few decades , especially 
 in lakes from glaciated areas .
the increase of solutes in lake water 
 was attributed to an enhanced weathering resulting from climate warming . until now ,
very little research has examined how changes in the 
 mountain cryosphere affect downstream water quality , including the 
 threats to ecosystem health and human use .
recent studies that investigated water quality in alpine watersheds 
 draining mineralized areas with sulfide - bearing lithologies demonstrate 
 that retreating glaciers and degrading 
 permafrost may adversely affect downstream 
 ecosystems receiving meltwater runoff .
the exposure of fresh surfaces 
 of sulfide - rich rocks to air and oxygenated water by retreating glaciers 
 or degrading permafrost increases the oxidation of sulfide minerals , 
 which is responsible for the generation of natural acid rock drainage 
 ( ard ) .
ard typically produces acid - sulfate waters enriched in fe , 
 al , mn , and various trace elements such as cu , ni , pb , zn , and others .
concentration of the dissolved minor and trace elements in natural 
 ard systems generated by retreating glaciers or degrading permafrost 
 can be comparable to ard associated with mining activity .
some of the mobilized trace elements associated with ard pose a 
 potential hazard to environmental quality and human health .
although ice loss in permafrost can be orders of magnitude slower 
 than in a glacier , the total permafrost area usually exceeds the glacier - covered 
 area in mountain environments and can contribute substantially to 
 the hydrology of alpine catchments .
for 
 example , the perennially frozen area in the european alps is approximately 
 three times larger than the glacier - covered area . despite the fact that rock glaciers are one of the most 
 common geomorphological expressions of mountain permafrost ,
still little is known about the geochemical content , especially 
 about the trace - element composition of supra- and subpermafrost meltwater 
 that drains rock glaciers . until recently
, 
 most of the research conducted on rock glaciers primarily focused 
 on their movement , origin , internal structure , and hydrology .
however , the impacts of rock glacier solute fluxes on lakes downstream 
 still remain largely obscure even though 
 more than half of the world population relies on fresh water from 
 mountain areas .
unexpected high 
 nickel and manganese concentrations exceeding the 
 appropriate eu limits for drinking water by more than an order of 
 magnitude have recently been reported in a study investigating temporal 
 trends in water chemistry of a remote high - mountain lake , lake rasass , 
 situated at the toe of an active rock glacier the european alps , italy .
the high concentrations of mn and ni were observed 
 in the lake water enriched in sulfates ( more than 4400 equivl ) .
in contrast , negligible concentrations of these 
 metals were recorded in the adjacent pond without a rock glacier in 
 the catchment . in an earlier study ,
application 
 of paleoecological techniques provided strong evidence of acidic conditions 
 in lake rasass ( ph 5.86.5 ) during at least the past two centuries .
the overall results of the previous studies 
 focused on the lake give evidence of naturally occurring ard generated 
 by the rock glacier in the lake rasass catchment .
the present 
 study aims to explore the effects of rock glacier solute 
 fluxes on a high alpine lake within a crystalline - rock watershed . 

we applied comparative research design in the study of two neighboring 
 water bodies , lake rasass and the adjacent pond , with and without 
 a rock glacier in their mineralized catchments having similar soil 
 conditions and underlying geology .
in addition , we used chironomid - based 
 paleolimnological techniques to track long - term temporal trends in 
 the ecotoxicological state of both water bodies and to establish their 
 baseline conditions .
depth = 9.3 m ) and the adjacent pond ( rpd ; surface area = 0.08 
 ha , max .
depth = 1.6 m ) are remote , fishless water bodies situated 
 just 50 m away from each other , above the actual and historical timberline 
 in the upper vinschgau valley ( 2682 m a.s.l . ) in the central eastern 
 alps , italy ( figure 1 ) .
the bedrock geology 
 of the valley belongs to the austroalpine tztal - stubai crystalline 
 complex , and the average mineral composition 
 of the bedrock is quartz ( 50% ) , feldspar ( 27% ) , muscovite ( 15% ) , chlorite 
 ( 6% ) , and dolomite ( 2% ) .
the chemical 
 composition of the bedrock is characterized by sources of disseminated 
 water - soluble sulfate ( 180 g g dry 
 weight ( dw ) ; total sulfur is 590 g g dw ) and metals , including trace elements mn ( 597 g 
 g dw ) , ni ( 24 g g dw ) and zn ( 90 g g dw ) .
the concentrations of mn and zn are comparable 
 with their earth crustal abundances , while 
 ni content is more than three times lower .
soil cover is sparse and 
 bedrock is exposed in 80% of the ras and rpd catchment areas . 

the active rock glacier extends on a north - facing slope down to the 
 shore of ras and occupies 18.5% of its catchment area which 
 adjoins the permafrost - free catchment of rpd .
both water bodies lacking 
 inflow streams have well - developed outflows . near the rpd shoreline , 
 there are visible sources of groundwater that infiltrates onshore 
 discharges .
since the rock glacier is located only a few meters upslope 
 from ras , its internal drainage system , a common feature of rock glaciers , is most likely connected to the 
 lake , and meltwater seeps from the rock glacier into ras through cracks 
 and fractures in the bedrock .
map showing the location of the study site ( a ) 
 and photograph of 
 lake rasass ( ras ) and the adjacent pond ( rpd ) and their catchments 
 ( b ) .
the map of italy is reproduced from http://d - maps.com/. since 1980 , summer and cold - season 
 ( october  may ) temperatures 
 in the european alps show a simultaneous , strong increase , which is 
 unprecedented over the last millennium . a multifold increase in concentrations of the dominant ions ( mg , so42 + , and ca ) and electrical conductivity in the ras water 
 over the last two decades ( 19862005 ) , recorded by thies et 
 al .
, may reflect current permafrost degradation 
 in the rock glacier under the recent warming .
monnier and kinnard assume that indications of degrading permafrost 
 in a rock glacier may also be the near - melting - point ( 0 c ) thermal 
 regime of permafrost year - round and a high water content ( > 40% ) 
 in 
 deeper parts of a rock glacier .
as solutes concentrate and the freezing 
 point decreases during fluid freezing , it is most likely that highly concentrated salt solutions seep from 
 the rock glacier into ras through the cold season .
both water bodies were sampled for 
 limnochemical analyses at least twice during the open - water seasons 
 of 20102012 .
water samples were taken with a patalas - schindler 
 sampler ( uwitec , austria ) at 2-m intervals along a vertical profile 
 in ras and from 0.5 m water depth in the shallower rpd .
the physicochemical 
 parameters of water , such as conductivity , ph , alkalinity , and concentrations 
 of major ions and nutrients were determined following standard methods 
 and analytical quality control procedures as described in mosello 
 and wathne . metals ( ag , al , as , ba ,
be , 
 cd , co , cr , cu , fe , mn , ni , pb , sr , ti , v , zn ) in water samples were 
 assayed by inductively coupled plasma - optical emission spectrometry 
 ( icp - oes ) with standard addition methods .
sampling strategy was designed to compare metal concentrations 
 in surface sediments and biota of the water bodies as follows : the 
 ras and rpd shallow waters against the ras deep water , and between 
 shallow waters of ras and rpd . to provide background information for 
 the sampling in the relatively deep ras
, bathymetric distribution 
 and substratum associations of the most abundant macroinvertebrates 
 were studied in the lake along a depth transect ( supporting information ( si ) , figure s1 ) .
twenty macroinvertebrate 
 samples were collected with an ekman grab sampler ( in deep water ) 
 and a kick net ( in the upper littoral ) at 0.51.0 m depth intervals 
 along the east  west transect across the deepest point in the 
 lake .
surface sediment ( 01 cm ) samples were collected using 
 an ekman grab sampler with a lining of plastic inside from three random 
 sites in shallow waters ( 0.51.5 m depth ) of both water bodies 
 and in deep water ( 68 m depth ) of ras .
the sediment and biota 
 sampling for subsequent multielement analysis was conducted in the 
 early open - water season of 2012 . for multielement analysis of 
 body tissues of aquatic organisms , larvae of the chironomid midge pseudodiamesa nivosa ( diptera : chironomidae ) ,
the oligochaete tubifex tubifex ( oligochaeta : tubificidae ) , and the aquatic 
 moss warnstorfia exannulata ( hypnales : amblystegiaceae ) 
 were collected with a biological bottom dredge ( efe & gb nets , 
 uk ) in the ras deep - water habitats ( 68 m depth ) .
larvae of 
 the chironomid p. nivosa were also collected in rpd 
 ( 0.51.5 m depth ) .
the predatory aquatic beetle agabus 
 bipustulatus ( coleoptera : dytiscidae ) was collected with 
 a kick net in shallow - water habitats ( 0.51.5 m depth ) of both 
 water bodies .
invertebrates were held in lake water at field temperatures 
 for transport to the laboratory where they were sorted according to 
 species , cleaned with distilled water , placed in petri dishes and 
 kept at 4 c for 24 h in order to purge the guts .
thereafter 
 they , as well as the aquatic moss w. exannulata , 
 were rinsed with distilled water and frozen pending analyses . only 
 plastic storage vessels and tools ( tweezers , scapulae , etc . ) were 
 used .
all plastic labware was soaked in 1% nitric acid for at least 
 24 h and rinsed with doubly deionized water to leach out metal ions .
all sediment and biota samples were dried 
 in an oven at 25 c 
 to a constant weight ( 34 days ) and homogenized with an agate 
 mortar and pestle , and triplicate 0.5 g aliquots of every sample were 
 used for the analysis .
the samples were dissolved with concentrated 
 nitric acid using the microwave digestion 
 technique .
a total of 42 elements ( ag , 
 al , as , ba , bi , br , ca , cd , cl , cr , cu , fe , ga , ge , hf , i , k , mg , 
 mn , mo , na , nb , ni , p , pb , rb , s , sb , se , si , sn , sr , ta , th , ti , 
 tl , u , v , w , y , zn , zr ) were assayed by energy dispersive x - ray fluorescence 
 ( edxrf ) analysis using calibration models based on fundamental parameter 
 methods . in the sediment samples ,
ten major elements , si , al , fe , 
 mn , mg , na , ca , k , ti , and p , were measured as their oxides .
analytical 
 accuracy was verified by the use of ccrmp ( canadian certified reference 
 materials project ) reference samples .
sediment cores 
 were taken using uwitec gravity and piston corers ( uwitec , austria ) 
 in the deepest point of both water bodies in the summer of 2011 .
the 
 119.2 cm long sediment sequence from ras was sectioned contiguously 
 into 156 samples : at 0.220.50 cm resolution within the top 
 24 cm and at 1 cm resolution from 24 cm to the bottom .
the 15.5 cm 
 long sediment sequence from rpd was sectioned contiguously every 0.5 
 cm , resulting in a total of 31 samples .
chronological control for 
 the sediment sequences was provided by accelerator mass spectrometry 
 ( ams ) radiocarbon dates derived from terrestrial plant macrofossils : 
 six ams c dates from the ras sequence and four dates 
 from the rpd sequence .
the bayesian statistical method of the oxcal 
 software package version 4.2.1 was used 
 to produce age - depth models ( si figure 
 s2 ) . according to these estimates , the ras and rpd sediment cores 
 span time periods of 10  200 years and 3200 
 years , respectively .
the ages are expressed in calibrated years before 
 present ( cal yr bp = calibrated years before ad 1950 ) ( see si 1 for further details ) .
incidence of mouthpart abnormalities 
 in chironomid larvae was used as measures of toxic stress in the water 
 bodies .
occurrence of mouthpart deformities in chironomids is a sublethal 
 effect of larval exposure to environmental stress factors .
since mouthpart deformities may also be induced 
 by toxic elements and their compounds not included in routine chemical 
 analyses , they may better reflect environmental quality than chemical 
 analysis alone .
assessment of these morphological abnormalities offers 
 an effective bioindication of toxic stress in modern freshwater ecosystems and over previous time periods .
the mentum and other 
 mouthparts of chironomid larvae damaged during the cleaning and mounting 
 process usually have abrupt breaks that are clearly visible and easily 
 distinguishable from deformed structures .
the chironomid - based paleolimnological study was designed 
 to track and compare past changes in incidence of larval mouthpart 
 deformities in identical chironomid taxa from ras and rpd . to provide 
 background information on the structure of the subfossil chironomid 
 assemblages ,
sediment core samples from both water bodies were analyzed 
 for species composition of the assemblages following standard procedures 
 outlined in brooks et al .
larval head capsules 
 of the two most common chironomid taxa , pseudodiamesa nivosa and micropsectra radialis - type , found in both dredge 
 and sediment core samples ( live specimens and their subfossil remains , 
 respectively ) from ras and rpd ( si figures 
 s1 and s3 ) were inspected for deformities of the mentum , a mouth part 
 which is typically well preserved in subfossil material .
three 
 biometric variables , head length , head width , and mentum 
 width , were measured for each head capsule .
these variables were used 
 to assign each head capsule to a specific larval stage of development 
 ( instar ) . a minimum of 100 third- and fourth - instar head capsules 
 of each of the two chironomid taxa , p. nivosa- and m. radialis - type , were inspected for mentum deformities 
 in each sediment sample at 250400 magnification using 
 a compound microscope .
the severity of mentum deformities was categorized 
 into three classes according to lenat : class i  slight deformities , such as fused 
 teeth ; class ii  more conspicuous , moderate deformities , such 
 as extra teeth , missing teeth , large gaps , and distinct asymmetry ; 
 and class iii  severe deformities , including at least two class 
 ii characteristics .
head capsules of live larvae were treated and 
 inspected for mentum deformities in the same manner as their subfossil 
 remains .
one - way analysis of variance ( anova ) , 
 followed by bonferroni post hoc test , was used to determine whether 
 there are any significant differences in the elemental composition 
 of water , sediment , and biota between the two adjacent water bodies , 
 as well as between the shallow and deep - water habitats of ras .
all 
 data were square root transformed to reduce the biasing effect of 
 extremely low or high element concentrations . the nonparametric mann 
whitney 
 u - test was used to detect significant differences in the frequency 
 of chironomid deformities between different time intervals in the 
 holocene .
chemical analysis 
 of water samples 
 revealed that the ratios of dissolved inorganic nitrogen ( din = no3-n + nh4-n ) to total 
 phosphorus ( tp ) exceed 100 by mass in ras and rpd ( see si 3 for more details ) .
this suggests that phytoplankton 
 communities in both water bodies are phosphorus - limited , defined by 
 bergstrm as din : tp > 3.4 by 
 mass . 

physico - chemical analysis of water taken from ras and rpd in 20102012 
 indicated that the striking differences in limnochemistry of these 
 two neighboring water bodies , recorded in 2005 , persisted to the present day ( table 1 ) .
although the water in both water bodies is dominated by the same 
 ions ( ca , mg , and so42 ) , the total ion concentration 
 and electrical conductivity in ras ( 10  500 equiv . 

l and 510 s cm , respectively ) are more than an order of magnitude greater than 
 in rpd ( 780 equiv .
the concentrations 
 of many dissolved minor and trace elements ( al , cu , mn , ni , sr , zn ) 
 are negligibly small ( one - way anova , bonferroni post hoc test , p < 0.05 ) in the circumneutral rpd ( ph 6.196.64 ) 
 compared to the acidic ras ( ph 4.935.86 ) ( table 1 ) .
however , 
 in the acidic ras , an extensive area of the lake bottom at 5.08.5 
 m water depth is coated with orange - colored iron oxyhydroxides ( si figures s1 ) .
it is known that under mildly 
 acidic conditions in surface waters affected by ard , the soluble ferrous 
 iron oxidizes to ferric iron , which precipitates as a ferric oxyhydroxide .
ras data for 20112012 , and 
 rpd data for 20102012 . for each parameter , values ( mean 
  se ) that share common letters ( k , m , or n ) do not differ significantly 
 ( one - way anova and bonferroni post hoc test , p < 
 0.05 ) .
values are for the 
 upper ( 05 
 m depth ) and lower ( 59 m depth ) parts of the ras water column .
na : not available , i.e. parameter 
 is not regulated by the european drinking water directive ; parameter values exceeding the appropriate european 
 union limit values for drinking water are shown in bold .
nh4-n = ammonium - nitrogen . in the ras catchment dominated by crystalline basement 
 rocks with 
 an inherently low acid buffering capacity , enhanced
cryogenic weathering 
 attributed to ice of the active rock glacier is the most likely mechanism 
 causing groundwater inputs of acidic , metal- and sulfate - enriched 
 water to the lake .
cryogenic weathering of the local bedrock , which 
 involves a plethora of physical , chemical and biological processes 
 ( ice segregation , freeze  thaw cycles , volumetric expansion 
 of water , hydration etc . ) , results in the exposure of fresh 
 bedrock surfaces to air and water in a complex rock glacier system 
 and leads to oxidation of sulfide minerals and the generation of ard . a solute - rich inflow sinks , as a rule , down the water column due 
 to its high density .
this effect can result in physical and chemical 
 stratification of the water column which reduces water circulation 
 and aeration of deep - water layers .
our data show that strong stratification 
 develops in ras during periods of prolonged ice cover ( 89 
 months ) .
after the ice breakup , the concentrations of major ions ( ca , mg , so42 + ) and minor and trace elements ( al , mn , ni , 
 sr , zn ) in the upper water layers ( 05 m ) are four to seven 
 times smaller than in the deeper waters ( 59 m ) , and the ph 
 decreases from 5.48 near the surface to 5.07 at depth ( table 1 and figure 2 ) .
depth profiles 
 of selected physical and chemical properties of 
 the ras water . electrical conductivity ( a ) , ph ( b ) , total ion ( c ) , 
 calcium ( d ) , magnesium ( e ) , and sulfate ( f ) profiles in the ras water 
 column on fourth july 2012 ( closed circles
; the lake was 70% 
 ice covered ) and 29th august 2012 ( open circles ; 1.5 months 
 prior to lake ice freeze - up ) .
thus , the short growing season in ras begins under extremely 
 unfavorable 
 conditions for aquatic life , especially in the deep water , and the 
 lake water may be dangerous for human consumption .
in fact , the concentrations 
 of manganese , nickel , aluminum , and sulfate in the deep water ( 59 
 m ) exceed the appropriate eu limits for drinking water 32 , 23 , 7 , and 2 times , respectively ( table 1 ) .
although solutes become more homogeneously distributed 
 in the water column by late august / early september due to destratification 
 by wind - induced mixing and convection ( figure 2 ) , concentrations of manganese , nickel , and aluminum along a water 
 depth gradient remain considerably higher ( 520820 , 140220 , 
 and 440620 g l , respectively ) than 
 the eu maximum permissible levels in drinking water .
total metal 
 concentration , however , does not necessarily correspond 
 with metal bioavailability and toxicity to aquatic organisms .
the proportion of the total metal content that 
 is available for uptake by biota depends greatly on the chemical form 
 in which the metals are present .
metals in aquatic systems can exist 
 in different chemical forms , including the dissolved inorganic complexes 
 with dissolved anions ( e.g. , hydroxides , sulfates , carbonates ) where 
 metals occur as free hydrated metal ions , organic complexes with dissolved 
 organic matter , and variety of particulate forms incorporated into 
 the matrix of solid organic or mineral particles .
the free ionic form of metals is the most relevant for 
 uptake by biota and hence the most toxic to aquatic life .
hydrogen ion activity ( ph ) and redox potential greatly affect metal 
 availability because of their strong influences on metal partitioning 
 and speciation .
numerous studies have shown that the free metal ion 
 concentrations increase with decreasing ph as well as upon oxidation 
 of initially reduced environments .
thus , both processes , namely winter stratification 
 resulting in 
 low ph in the deep waters ( ph 5.1 ) , and oxygenation of the 
 metal - rich hypolimnion and sediment during the open - water season , 
 may result in greater bioavailability and toxicity of metals to biota 
 in ras .
the concentrations of 
 most metals in the 
 sediments of ras and rpd are comparable with their abundances in the 
 earth s continental crust ( figure 3 ) . 

rather high concentrations of lead and zinc in sediments of both water 
 bodies may be attributed to the site - specific geologic conditions , 
 whereas elevated nickel and copper concentrations ( 244 and 179 g 
 g , respectively ) in the ras deep - water sediments 
 are likely to be caused by enhanced accumulation of fine - grained particles 
 in the deep water .
these particles provide a large surface area for 
 the sorption of metals at the water - sediment interface .
concentrations ( mean  se ; n = 3 ) 
 of copper 
 ( a ) , zinc ( b ) , lead ( c ) , nickel ( d ) , chromium ( e ) , aluminum ( f ) , iron 
 ( g ) , titanium ( h ) , manganese ( i ) , and vanadium ( j ) in surface sediments 
 and biota of rpd and the ras shallow and deep waters . for each metal , 
 means followed by the same letter are not significantly different 
 ( one - way anova , bonferroni post hoc test , p < 
 0.05 ) .
the abundances of the chemical elements in the earth s 
 continental crust are shown with green 
 lines .
concentrations 
 of 10 elements ( cu , zn , 
 pb , ni , cr , al , fe , ti , mn , and v ) were significantly higher ( one - way 
 anova , bonferroni post hoc test , p < 0.05 ) in 
 the larvae of pseudodiamesa nivosa , omnivores that 
 feed on a wide variety of foods from detritus to small invertebrates , collected from the ras deep water than from 
 rpd unaffected by the rock glacier solutes ( figure 3 ) .
this suggests a high bioavailability of these metals in 
 the ras deep - water habitats .
the 
 aquatic moss warnstorfia exannulata , which prefers 
 mildly acidic conditions , have colonized 
 an extensive area of the ras bottom at 58 
 m water depth ( si figures s1 ) . increased 
 concentrations of nickel and aluminum in the w. exannulata tissues were comparable to those recorded in the chironomid larvae 
 from the ras deep - water habitats ( figure 3 ) . 

they are tolerant of metal contamination , long - lived , 
 and have large cationic exchange properties within the cell wall . 

owing to the lack of roots , metal uptake occurs straight from the 
 water where they develop . among all benthic organisms 
 analyzed from 
 the ras deep water , the highest concentrations of cu , zn , and pb were 
 recorded in the body tissues of the sediment - ingesting oligochaete tubifex tubifex ( one - way anova , bonferroni post hoc test , p < 0.05 ) .
the cu concentrations in the t. tubifex tissues were 2.5 times greater ( one - way anova , bonferroni 
 post hoc test , p < 0.05 ) than that in surrounding 
 sediments ( figure 3 ) .
ingestion of contaminated 
 sediment has been shown to be the major source of particle - sorbed 
 metals to sediment - feeding organisms .
the cu concentrations in the predatory 
 beetle agabus bipustulatus , which is common in shallow - water 
 habitats of both water bodies , were also significantly higher ( one - way 
 anova , bonferroni post hoc test , p < 0.05 ) in 
 ras than those from rpd ( figure 3 ) .
results 
 of a recent study demonstrate that predatory 
 aquatic beetles are capable of reflecting trace elements bioaccumulation 
 in habitats with different disturbance levels , although their innate 
 ability to take up / excrete metals may vary widely between species .
inspection 
 of modern pseudodiamesa nivosa and micropsectra 
 radialis - type larvae , represented by live specimens and their 
 recent remains in surface sediments , for deformities of mentum revealed 
 the morphological abnormalities in the ras p. nivosa population .
no mentum deformities were noted upon inspection of p. nivosa from rpd and m. radialis - type 
 from both water bodies .
although mentum deformities in micropsectra larvae have been recorded at some metal - contaminated sites , it is likely that the p. nivosa larvae are more 
 susceptible to the harmful effects of acidic , metal - rich environments 
 than the m. radialis - type larvae .
incidence of mentum 
 deformities in the live p. nivosa larvae from ras 
 varied from 81% to 87% ( n = 3 ) and was comparable 
 with the frequency of mentum deformities among recent remains of p. nivosa in the ras surface sediments ( 7887% , n = 3 ) .
the observed deformities were categorized into two 
 classes following lenat : class i  fused 
 median tooth ( 3034% , n = 6 ) , and class ii  large 
 median gap ( 4855% , n = 6 ) or extra teeth 
 ( < 2% , n = 6 ) ( figure 4 ) . 

mentum abnormalities are generally related to a physiological disturbance 
 during the molting process , as a somatic response to a wide range 
 of environmental stress factors , but not to a genetic heritability 
 of larvae . in many polluted sites ,
the total incidence of deformities 
 in chironomid populations usually varies between 20% and 50% , but the incidence of abnormalities in the ras p. nivosa population ( 7887% ) was as high as that recorded in a population 
 of chironomus spp .
( 83% ) , another chironomid 
 taxon , in a site heavily polluted by radionuclides and trace metals .
micrographs ( 200 magnification ) of larval menta 
 of the chironomid pseudodiamesa nivosa from the ras 
 modern and subfossil material : 
 normal mentum ( a ) and deformed menta ( b  e ) .
the severity of 
 the observed deformities was categorized into two classes following 
 lenat : class i ( slight deformities)fused 
 median tooth ( b , c ) , and class ii ( more conspicuous deformities)large 
 median gap ( d ) and extra teeth ( e ) .
the analysis of chironomid remains 
 in core sediment samples showed 
 that a high total incidence of p. nivosa mentum deformities 
 of 70% persisted in ras during the last 2.5 millennia and 
 was significantly lower ( mann  whitney u - test , p < 0.0004 ) , around 40% , in the early holocene ( 10  1509800 
 cal yr bp ) ( figure 5 ) .
the incidence of the 
 class ii mentum deformities ( median gap or extra teeth ) in p. nivosa from ras was also significantly higher ( mann  whitney 
 u - test , p < 0.0008 ) in the late holocene ( 49% ) 
 than in the early holocene ( 21% ) .
unfortunately , the cold - stenothermal p. nivosa was absent in ras , like other high alpine lakes , through a relatively warm interval of the early 
 and mid - holocene ( figure 5 , si figure s3 ) , and this species can not be used for reconstruction 
 of temporal trends in the ecotoxicological state of ras over the entire 
 holocene . in rpd , the frequency of p. nivosa mentum 
 abnormalities did not exceed 2% ( only the class
i mentum deformities 
 was recorded ) through the last 3.2 millennia , or , in other words , 
 it did not exceed the frequency of chironomid deformities found in 
 natural unstressed environments ( up to 1214% ) .
relative 
 abundance of pseudodiamesa nivosa in 
 chironomid assemblages and the total incidence of p. nivosa mentum deformities in the sediment records from rpd ( a ) and ras 
 ( b ) , and holocene july air temperatures ( tjuly ) reconstructed for the eastern alps ( c ) .
the results provide evidence that 
 the present adverse conditions 
 in the lake are not unprecedented in its holocene history and can not 
 be associated exclusively with global warming and enhanced thawing 
 of the rock glacier in recent years .
it is likely that the development 
 of the rock glacier started at the onset of the holocene , when the 
 area occupied by the modern rock glacier became deglaciated .
the formation 
 of ras at the toe of the rock glacier most probably began at the onset 
 of acidic drainage in the freshly deglaciated area .
overall , 
 our findings illustrate that natural ard systems generated 
 by active rock glaciers in mineralized areas with sulfide - bearing 
 lithologies may seriously disturb lake ecosystems and endanger the 
 quality of drinking water in pristine alpine environments .
more adverse 
 effects may be expected in lakes situated at the toe of greater and 
 more active rock glaciers than the one in the ras catchment .
 despite 
 the fact that rock glaciers are one of the most common 
 geomorphological expressions of mountain permafrost , the impacts of 
 their solute fluxes on lakes still remain largely obscure . 
 we examined 
 water and sediment chemistry , and biota of two neighboring water bodies 
 with and without a rock glacier in their catchments in the european 
 alps . 
 paleolimnological techniques were applied to track long - term 
 temporal trends in the ecotoxicological state of the water bodies 
 and to establish their baseline conditions . 
 we show that the active 
 rock glacier in the mineralized catchment of lake rasass ( ras ) represents 
 a potent source of acid rock drainage that results in enormous concentrations 
 of metals in water , sediment , and biota of ras . 
 the incidence of morphological 
 abnormalities in the ras population of pseudodiamesa nivosa , a chironomid midge , is as high as that recorded in chironomid populations 
 inhabiting sites heavily contaminated by trace metals of anthropogenic 
 origin . 
 the incidence of morphological deformities in p. nivosa of 70% persisted in ras during the last 2.5 millennia and 
 was 40% in the early holocene . 
 the formation of ras at the 
 toe of the rock glacier most probably began at the onset of acidic 
 drainage in the freshly deglaciated area . 
 the present adverse conditions 
 are not unprecedented in the lake s history and can not be associated 
 exclusively with enhanced thawing of the rock glacier in recent years . 