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Toxicity of inorganic aluminium at spring snowmelt—In-stream bioassays with brown trout (Salmo trutta L.)
Science of the Total Environment 437 (2012) 422–432
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Toxicity of inorganic aluminium at spring snowmelt—In-stream bioassays with brown trout (Salmo trutta L.) Cecilia M. Andrén a,⁎, Emil Rydin b a b
Dept. of Applied Environmental Science, ITM, Stockholm University, S-106 91 Stockholm, Sweden Erken Laboratory, Dept. of Ecology and Genetics, Uppsala University, S-761 73 Norrtälje, Sweden
H I G H L I G H T S ► ► ► ► ►
Brown trout were exposed in situ to a gradient of pH and Ali in humic streams. pH above 5.0 and Ali below 20 μg/L sustain healthy brown trout populations. Low pH resulted in a tendency to decreased blood chloride levels. High Ali increased gill Al and haemoglobin suggesting that respiration was effected. The thresholds can be used to support liming strategies in recovering freshwaters.
a r t i c l e
i n f o
Article history: Received 13 March 2012 Received in revised form 31 July 2012 Accepted 1 August 2012 Available online xxxx Keywords: Acidification Brown trout Thresholds Ali & pH Gill accumulation Al Blood physiology Liming strategy
a b s t r a c t Although the acid load has decreased throughout Scandinavia, acidic soils still mobilise aluminium (Al) that is harmful to brown trout. We hypothesise that there are thresholds for Al toxicity and that the toxicity can be traced from the water content to gill accumulation and the consequential physiological effects. During snowmelt, yearlings were exposed to a gradient of pH and inorganic monomeric Al (Ali) in humic streams to study the toxic effects and mortality. Gill Al and physiological blood analyses [haemoglobin (Hb), plasma chloride (P-Cl) and glucose (Glu)] were measured. As the water quality deteriorated, Al accumulated on the gills; Hb and Glu increased; P-Cl decreased, and mortality occurred. Moribund fish had significantly increased gill Al and Hb, suggesting that respiratory disturbances contributed to mortality. Decreased P-Cl and plasma availability indicated an ion regulatory disturbance and possibly circulatory collapse. Ali should be less than 20 μg/L, and pH higher than 5.0, to sustain healthy brown trout populations. These thresholds can be used to fine-tune lime dose, as both Ali and pH levels have to be balanced to prevent harm in the recovering aquatic biota. Although Al is tightly linked to pH, local variation in Al availability in soil and bedrock affects the Al release and subsequent toxic Ali episodes in some catchment areas. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Freshwater bodies in base-poor areas in Europe and North America are recovering chemically from acidification (Skjelkvale et al., 2005; Stoddard et al., 1999) due to decreased sulphur (S) deposition. In areas with deep soil layers, acidification effects might be prolonged as the soil stores S. Many soils still have a large store of organic S, and differences in the rate of decrease between S deposition and runoff cause a considerable time lag, with recovery delayed for decades
⁎ Corresponding author. Tel.: +46 73 707 89 99; fax: +46 8 674 76 36. E-mail addresses: [email protected] (C.M. Andrén), [email protected] (E. Rydin). 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2012.08.006
(Morth et al., 2005; Wright et al., 2005). Furthermore, climate change disturbs runoff patterns and creates hydrological episodes (Arnell, 1999; Bergstrom et al., 2001). These episodes mobilise S and cause acid surges that are harmful to biota (e.g. Kroglund et al., 2008; Lepori and Ormerod, 2005; McCormick et al., 2009). Few records of biological recovery have been documented (Wright et al., 2005), except in Norwegian clear waters (e.g. Raddum et al., 2001) with catchments draining bedrock with shallow soil layers, which may recover faster than areas with deeper soil layers. In North American field studies on brook trout (Salvelinus fontinalis), similar mortality was recorded for 2001–2003 as in previous studies (1984–1985, 1988–1990, and 1997), suggesting no improvement in water quality (Baldigo et al., 2007). Soil aluminium (Al) is dissolved and solubilised in water by low pH, generating inorganic monomeric Al (Ali), which is highly toxic to fish (Driscoll and Schecher, 1990; Gensemer and Playle, 1999). The mere presence of Ali in surface waters has been argued as an
C.M. Andrén, E. Rydin / Science of the Total Environment 437 (2012) 422–432
unambiguous indication of anthropogenic acidification (Lawrence et al., 2007). Fish death in acidic Al-rich water can be caused by several dysfunctions: ion regulatory disturbances (a net loss of plasma electrolytes and a net gain of plasma-H+ and -Al); respiratory dysfunction (concurrent hypoxia, hypercapnia and plasma acidosis); and osmoregulatory breakdown (a net influx of water) (Exley et al., 1991). Al accumulates in the alkaline microenvironment of the gill, leading to tissue damage (Muniz and Leivestad, 1980; Playle and Wood, 1989) that reduces the oxygen and carbon dioxide exchange (Wood and McDonald, 1987) but also disturb ion and osmoregulation. However, pH toxicity has no effect on gas exchange but primarily disturbs ion regulation (inhibits transcellular Na + and Cl − uptake) and osmoregulation (opens paracellular channels) (Wood and McDonald, 1987). Brown trout (Salmo trutta L.) is less vulnerable to acidity than salmon (Salmo salar L.) (Poleo et al., 1997) and is a key goal for lime treatment in Sweden (SEPA, 2010). To plan and balance the lime dose, given that Al availability varies locally, knowledge regarding the environmental effects by concomitant Al-pH toxicity is required. In this study, we expose brown trout to a naturally occurring Ali and pH gradient in humic streams to determine when Ali and pH become toxic to yearling brown trout. We trace the Al effect via accumulation on the gills and the consequential physiological effects. Furthermore, we aimed at distinguishing the Al toxicity from the pH toxicity via the physiological effects.
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2. Materials and methods 2.1. Study area The study was performed in humic streams on coniferous hills located in a base-poor area in the middle of Sweden, where the acid deposition is quite low (Fig. 1). The catchments' buffering capacities are low with granite and gneiss bedrock overlain by a thin layer of till. At springtime in this region, the snow melt flood decreases the pH and increases Ali; although the water's humic content might moderate the Ali toxicity. All streams had minor wetlands upstream of the exposure sites in addition to forest (Table 1). The streams are pristine besides some foresting activities; however, downstream of the Havssvalgsbäcken site, lime was spread before 2000. This lime could have accumulated in the lake 2.5 km upstream the site in Örvallsbäcken and might still influence this stream when strong undercurrents re-suspend and dissolve the lime from the sediment. The streams were chosen based on known water quality to obtain a gradient for the trout exposures: from acidic to slightly acidic to neutral conditions during the snowmelt. All streams have or have had native brown trout populations, with densities ranging from 0 to 77 trout/100 m 2 estimated by electrofishing autumn 2002 (Table 1). In the headwaters (Havssvalgsbäcken), the brown trout population was first diminished by wetland trenching until the fish apparently vanished by acidification and liming began in the early eighties. The
o 4065
Lat. 61°43' Long.16°40' o 4125
o 4250
4041 o
4070 o
o
4081
Fig. 1. Catchment areas and stream exposure sites, with forest (green), wetland (brown), brooks and Lake Örvallssjön (blue). The inserted map shows the location of the study area in Sweden.
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Table 1 Major characteristics of the six stream cage sites. Brooks (site)
Catchment area (km2)
Altitude (m a.s.l.)
Length (km)
Örvallsbäcken (4250) Stängmyrbäcken (4065) Lövåsbäcken (4125) Brånvallsbäcken (4041) Häståbäcken (4081) Havssvalgsbäcken (4070)
6.6 4.8 4.8 4.3 4.7 2.0
114 111 160 153 263 317
6.5 6.7 6.3 3.3 6.9 2.2
a
Land use Forest
Wetland
Water
Other
86% 97% 92% 83% 80% 74%
12% 2% 8% 17% 20% 26%
2% 0% 0% 0% 0% 0%
0% 1% 0% 0% 0% 0%
Populationa trout/100 m2 19 26 77 22 22 0
Brown trout density electro fishing 2002.
mires have now mended and the water quality has improved and a few adult trout have been observed since 2009; though prior to 1940, these were some of the best trout waters in the area according to local fishermen.
2.2. Fish rearing For the exposures, brown trout yearlings (S. trutta L.) (mean 11 cm and 14 g) were used from a nearby hatchery (Bogården fish farm in Ljusnan) also humic but more neutral water quality (pH c.7 and TOC c. 16 mg/L). A local strain, Mellan–Ljusnan, was stocked at all sites at the start of the trials (60 trout/cage) and restocked later in smaller quantities when necessary. This strain was similar to indigenous stream strains and was not expected to have physiological responses caused by differences in water quality at the site of rearing (McWilliams, 1980). Control samples of gills were taken twice (6 + 6 trouts) when the fish were stocked in the cages.
2.3. Fish handling The fish were kept in cages made of stainless steel drums from top loading washing machines, c. 50 L holding 60 trouts i.e. 13.2 kg/m 3. These cages enabled free water passage without allowing the fish to escape and were stable against the stream current. They are commonly used for storing small fish as bait for fishing. The fish cages were checked at each water sampling (see Section 2.5, twice a week and in the acid stream daily). Dead fish were removed, and the cage positions were adjusted to ensure sufficient water flow and avoiding stressful eddies. All fish handling was performed with care according to the study licence (C75/2 2002) from the Uppsala Animal Testing Ethical Council at Tierp District Court.
2.4. Fish sampling Each stream exposure site was visited as often as possible to collect water and fish samples at the same time. The plan was to sample twice a week or more based on apparent fish responses. One stream required more or less daily checks, while the other five were sampled according to the plan. Groups of six trout with normal behaviour were chosen for sampling, but also fish with altered behaviour were collected. For each individual fish, potential altered behaviour was noted, e.g., if they moved slowly, barely breathed or swam upside down, and these moribund fish were designated as affected (as opposed to fit and normal). For comparison, gills from a few dead fishes were also sampled to compare gill-Al with fishes with altered behaviour. Gill-Al data from dead fish is uncertain and should be used carefully. Fish were anesthetised by a blow to the head. Blood samples were taken from the caudal vein after the tail was cut with a sterile scalpel. After obtaining a gill arch sample, the fish were killed by mechanical destruction of the brain.
2.5. Fish analysis Haemoglobin (Hb) and glucose (Glu) were determined on site with Hemocue microcuvettes and spectrophotometers (Hemocue, Ängelholm, Sweden). The remaining blood sample was centrifuged at 2000 ×g for 5 min within 15 min of sampling. The supernatant plasma was removed by pipetting and preserved by freezing; plasma-chloride (P-Cl) was determined with a Radiometer CMT-10 chloride titrator (Radiometer, Copenhagen, Denmark). The second gill arch on the left side was excised from the fish and frozen in a pre-weighed, acid-washed plastic bottle. The gill arch was freeze-dried, weighed and digested in 10% HNO3. The Al in the gill (gill-Al) was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES), and the results were reported as μg/g gill dry weight (dw). 2.6. Water sampling and analysis Water samples were collected twice weekly except in the most acid stream where daily samples were collected at high flow. Samples were taken in polythene bottles that were rinsed with stream water, submersed below the surface and filled to the brim; the cap was screwed on tightly without air in the bottle. Separate bottles were used for the different analyses which were performed with standard analytical methods following quality assurance and control procedures (within the laboratory accreditation from SWEDAC following ISO/IEC 17025). Four variables were used to characterise the water quality. The pH was measured in un-aerated samples with a glass combination electrode (Radiometer). The Al fractions were measured within 1 day as the total monomeric Al (Alm) and organic monomeric Al (Alo) (Driscoll, 1984) by the colorimetric reaction of pyrocatechol violet (Dougan and Wilson, 1974) without acid addition combined with cation exchange using continuous flow analysis with an AutoAnalyzer (Technicon Corp., Tarrytown, New York, USA) at 580 nm. The Alo passed through the column, while the potentially toxic fraction, Ali, was retained by the cation exchange resin (and hence could be calculated as the difference between Alm and Alo). The analytical method for inorganic Al used here exclusively detects monomeric Ali, low molecular mass Al (i.e., Al 3 +, Al(OH)2+, Al(OH) 2 +, AlF2+, AlF 2 +, Al(SO4) +), not including high molecular mass Al polymers (Andrén and Rydin, 2009). The variability for the Ali duplicate samples expressed as relative standard deviation was below 10%, and the detection limit was 3 μg/L. Values below this limit were given the nominal value of 2 μg/L; however, when the Alo exceeded the Alm, the Ali was set to 0 μg/L. The total organic carbon (TOC) was determined by the carbon analyser Shimadzu TOC5000, and calcium was determined with flame atomic absorption spectrometry (AAS). 2.7. Experimental conditions The brown trout were exposed between 21 March and 26 April 2002. In this year, the snowmelt and spring flood were quite moderate
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(discharge 20–670 L/s) after an initial melt when the fish were stocked. The temperature increased in the water from 0 °C to 7.8 °C as spring advanced with lowest temperatures found at the acid site (0.8º ±0.5 °C) which was located at the highest elevation (317 m.a.sl.). Yet a wide spectrum of conditions was covered among the sites (Table 2), with pH values ranging from 4.6 to 6.8 and Ali ranging from 0 to 70 μg/L. There were no confluences between water of different qualities (such as acid and more neutral or limed water) nearby the cage sites so the experiment was conducted in fairly stable waters not affected by mixing zones. Still a test was done at the sites prior to the study that showed marginal difference between field fractionation and lab fractionation within 24 h (unpubl data). The fluctuations in pH and Ali were considered to mainly be caused by snow melting and variances in discharge. The maximum exposure period was 9 days at the acid site and 37 days (the entire melting period) at the other sites. Therefore in order to follow the conditions throughout the snow-melt the acid stream was restocked five times as the trouts then either had died or were spent for sampling. There was no significant separate effect of the exposure time on mortality or any other fish parameter beyond that of the site (two-way ANOVA factors: site and exposure length b9 days or >9 days, P b 0.01).
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3. Results 3.1. Fish response All fish survived in five of the six streams even if their exposure time covered the entire snowmelt period (37 days). In the acid stream though, mortality varied between 10 and 100% for five consecutive rounds of exposure (maximum exposure length 9 days) and was associated with low pH and elevated levels of Ali (Fig. 2). The overall water quality encompassed a wide pH (4.6–6.8) and Ali range (0–69 μg/L) and fluctuated both over time and between sites. The Ali varied with pH (r 2 = 0.80, n = 73, P b 0.001), and when the pH dropped, the Ali increased. The gill-Al in the fish sampled prior to the exposure was (8 ± 7 μg/g dw, n = 12) and represents the background levels in the fish. When the fish were exposed, the accumulated amount of gill-Al (3–264 μg/g dw) and the variation (s.d. 40 μg/g dw) was higher than the background levels. The gill-Al was correlated with the Ali and pH (r 2 = 0.44 and 0.42, respectively, n = 73, P b 0.001). The Hb (66–153 g/L) was also correlated with the Ali and pH (r 2 = 0.47 and 0.57, respectively, n = 62, P b 0.001). The Hb showed high variation (s.d. 16 g/L) with the highest levels (115–153 g/L) found in the fish from the most acidic and Ali-rich stream. Lower Hb levels (66–129 g/L) with less variation (s.d. 10 g/ L) were noted for the fish exposed to more neutral streams. In contrast to Hb the levels of P-Cl (30–110 mmol/L) accompanied pH development; low pH corresponded to lower levels of P-Cl (correlation to Ali, r 2 = 0.59 and pH, r 2 = 0.69, n = 60, P b 0.001). Glu levels (2.2–18.3 mmol/L) were consistently higher at low pH and high Ali (correlation to Ali, r 2 = 0.61 and pH, r 2 = 0.70, n = 62, P b 0.001). The accumulated gill-Al was in turn correlated to increased Hb and Glu and decreased P-Cl (Hb, r 2 = 0.26; Glu, r 2 = 0.36; P-Cl, r 2 = 0.44; n = 62, 62, and 60, respectively, P b 0.001). The effects of stream water conditions on fish response were more apparent when grouped by exposure (n = 6 individuals/sampling) and ordered by decreasing pH (Fig. 3). In the acid stream, fish had more Al accumulated on the gills and showed higher Hb levels. P-Cl decreased and Glu increased in the more acidic water compared to neutral conditions. The repeated rounds of exposure at the acid site showed consistent results. All exposures fell into three categories based on water quality: neutral, slightly acidic and acid water (ANOVA Tukey HSD, P b 0.001) defining the different water qualities of the exposures based on the variables pH and Ali. This division into three categories resulted in nearly equal numbers of observations per category; neutral (18, 24), slightly acidic (24, 28) and acid (18, 21) for blood physiology and gill-Al respectively, thereby enhancing the statistical relevance. For the fish in all three exposure categories, the gill-Al, P-Cl and Glu displayed significant differences (ANOVA Tukey HSD, P b 0.001). For the Hb, only the acidic water was strongly significantly different from the other two categories (P b 0.001) and weakly significantly different (P b 0.05) between the neutral and slightly acidic waters.
2.8. Fish mortality The mortality was calculated in two ways, proportional (%) and categorical (0/+). The proportional mortality was calculated as the share of dead fish out of exposed fish (adjusted for fish removed for sampling) i.e., the total number of dead fish at the site divided by the number of alive fish left at the site. The observed death or accumulated categorical mortality (denoted with crosses in contrast to healthy fish denoted with o), was used for groups of fish (n = 6) and defined as if any mortalities had occurred for this exposure (stocking per stream) since the start of the exposure. 2.9. Statistical analyses The results were divided into different scales to make the study more transparent: individual fish [gill-Al (n = 446), Hb (n = 370), Glu (n = 362), and P-Cl (n = 290)], grouped (six fish from each sampling occasion, n = 73) or separate rounds of exposures (i.e., stockings/stream; 5 streams were stocked once, and there were 5 consecutive stockings in the acid stream, n = 10). Statistics was calculated with SPSS for Windows release 15.0.0 (SPSS Inc., Chicago, Illinois, USA) and presented as average, standard deviation (s.d.) and range (minimum–maximum value). Several statistical tests were performed, including Pearson correlation analysis, analysis of variance (one-way and two-way ANOVA) with the Tukey–Kramer Honestly Significant Difference (HSD) tests and one-sided t-tests was used to describe the relationship between the water and fish measurements. To clearly illustrate the link between water quality and fish health, scatter and box plots were used.
Table 2 Water quality in the six stream cage sites for the exposure periods. Brooks (site) Örvallsbäcken (4250) Stängmyrbäcken (4065) Lövåsbäcken (4125) Brånvallsbäcken (4041) Häståbäcken (4081) Havssvalgsbäcken (4070)
pH
Ali (μg/L)
TOC (mg/L)
Ca (meqv/L)
Median
Min
Max
Median
Min
Max
Median
Min
Max
Median
Min
Max
6.4 6.3 6.3 5.5 5.4 4.8
6.2 6.2 6.0 5.3 5.0 4.6
6.7 6.7 6.8 6.2 6.2 5.0
5 6 3 19 26 49
0 0 0 5 4 18
7 12 17 34 44 69
14 11 13 16 16 18
13 9 10 14 12 15
17 12 14 18 18 19
0.14 0.08 0.10 0.07 0.09 0.06
0.11 0.08 0.08 0.07 0.06 0.04
0.19 0.10 0.21 0.09 0.14 0.06
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Site 4065
4125
4041
4081
4070
pH
Ali (µg/L)
Al (µg/g dw)
Hb (g/l)
P-Cl (mmol/L)
Glu (mmol/l)
Mortality (%)
4250
Date Fig. 2. Temporal variation of the variables observed during the spring flood in 2002 for the six streams. The pH and Ali (water) are at the bottom, followed by the Al accumulation (gills), Hb (blood), P-Cl (blood), Glu (blood), individual fish results, and the cumulative mortality at the top.
3.2. Water quality—fish status To scrutinise the effects on Al accumulation, physiology and fish status (health condition) data were divided into levels according to pH (0.2 intervals) and Ali (10 μg/L intervals) within the pH and Ali ranges (Figs. 4 and 5). Affected moribund fish (swimming erratically and hypo ventilating) had more Al accumulated on their gills compared to unaffected fish within the same Ali level (20–30, 30–40,
40–50 μg Ali/L, t-test, P b 0.001, and > 50 μg Ali/L, P b 0.01, Fig. 4). The Hb increased with the Ali below 50 μg Ali/L, with significantly higher levels for affected fish at the same Ali levels (t-test, P b 0.001); however, above 50 μg Ali/L, the Hb levels dropped. In two pH classes, more Al accumulated in affected fish compared to unaffected fish within the same level (4.6–4.8, 5.0–5.2, t-test, P b 0.001, Fig. 5), which can be expected as Ali is tightly linked to pH. There were too few cases (as affected fish had thickened blood and not
C.M. Andrén, E. Rydin / Science of the Total Environment 437 (2012) 422–432
b)
c)
d)
Hb (g/L)
Ali (µg/L)
a)
427
e)
f)
Fig. 3. Boxplots showing the exposure conditions and fish responses for each site and round of exposure (five consecutive rounds of exposures at the acid site are shadowed in grey); a) pH; b) Ali; c) Al accumulated on the gills; d) Hb; e) P-Cl; and f) Glu. Site numbers are shown below the x-axes and days of exposure above the x-axes. The panels' a–d and f all have data for the same ten boxes. Please note that not enough blood could be sampled to determine the P-Cl in the first exposure at the acid site so only nine boxes are shown in panel e. Lines in boxes are medians; the ends of boxes are quartiles; dots represent individual values, and whiskers show the range of values. Outliers (°) are defined as results outside the quartiles, while extremes (*) are more than three quartiles from the median.
enough plasma for Cl analysis) to statistically test the difference in fish condition for plasma-Cl. There was a trend of lower P-Cl in the affected fish as pH decreased (Fig. 5). Moribund fish with affected behaviour had comparable levels of accumulated Al compared with the dead fish (t-test, P b 0.001). 4. Discussion We showed that Ali occurred in detectable concentrations in humic boreal streams in central Sweden, an unambiguous indication of acidic deposition (Lawrence et al., 2007). Ali was harmful at the slightly acidic sites and deadly to brown trout at the most acidic site. Subsequently, the ongoing influence of suboptimal water qualities might be detrimental to present-day brown trout residents as
noted by the scarcity of trout at the acidic site (Table 1). The analysed Ali consisted only of monomeric forms, which were directly linked to toxic and mortality effects. Moreover, the Ali results showed significant correlations to pH and gill-Al, with no evident amelioration by the relatively high TOC levels (observed by e.g. Neville, 1985; Roy and Campbell, 1997). The strong link to both pH and gill-Al suggests that there was little or no effect of storage time on Ali concentration before analysis opposing previous suggestions in studies with less focus on Al chemistry (Laudon et al., 2005; Serrano et al., 2008). These inorganic monomeric cationic form of Al that can be detected with laboratory fractionation occurs in acidic water (as in this study) unlike the polymeric highly toxic and transient Al complexes found in mixing zones between acidic and limed waters (Poleo, 1995). In this study, we demonstrated toxicity from Ali and pH but
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a)
c)
300
150
P-Cl (mmol/L)
Al (µg/g dw)
250 200 150 100
100
50
50 0 <10
10-20
***
***
***
**
20-30
30-40
40-50
>50
0 N=102 <10
Ali range (µg/L)
63
42-1
38-1
11-10
11-11
*** 10-20
20-30
30-40
40-50
>50
10-20 20-30 30-40 40-50
>50
Ali range (µg/L)
b) 150
d) 20,0
Glu (mmol/L)
15,0
Hb (g/L)
100
50
10,0
5,0
0 <10
10-20
***
***
***
20-30
30-40
40-50
0,0 >50
*** <10
Ali range (µg/L)
Ali range (µg/L)
Fig. 4. Box plots with fish response and condition plotted against water quality (Ali split into ranges). Affected fish (black hatched bars) had slow movements, were barely breathing or swam upside down. Ali range and a) Al accumulated on the gills, b) Hb, c) P-Cl and d) Glu. Lines in boxes are medians, the ends of the boxes are quartiles, dots represent individual values, and whiskers show the range of values. Outliers (°) are defined as results outside the quartiles, while extremes (*) are more than three quartiles from the median. Statistically significant differences (t-test) are denoted by *** (P b 0.001) or ** (P b 0.01). Numbers of samples (N) are displayed for P-Cl, which had few observations in some ranges. All other boxes are based on at least six samples (one fish group sampling).
it was not possible to separate the toxic effects of low pH and high Ali; nonetheless, the results showed that both toxic agents are operative even though the waters were humic. Therefore, given that the need for lime is currently declining, efforts should address the combined effects of Ali and pH and not solely monitor the minimum pH to prevent damage to recovering sensitive trout populations and other biota. Still, only pH, alkalinity, Ca, Mg and colour are routinely measured and Ali is only determined occasionally as the analysis is considered complicated and costly. In our experiments, brown trout mortality was associated with elevated levels of Ali and reduced pH—a link observed for fish in the early days of acidification research (Cronan and Schofield, 1979; Dickson, 1978; Driscoll et al., 1980). The toxicity was, as expected, complex because pH and Ali have parallel and concurrent effects. The Ali level in the water was related to the burden of Al on the gills, as previously documented for other salmonids in streams (Lacroix et al., 1990) as well as in vitro (Playle and Wood, 1989) or by controlled experiments (e.g. Kroglund et al., 2001). The gill-Al in turn affected the physiology related to the Hb, P-Cl and Glu. Separate toxic effects of increased Ali and decreased pH levels could not be differentiated by fish mortality. Combining fish condition with fish physiology through various data clusters, we could partially detect the different effects from pH and Ali. Researchers using salmon (Neville and Campbell, 1988), brown trout fry (Sayer et al., 1991) and brown trout in mixing zones (Witters et al., 1996) have documented concomitant Al and pH effects, with Al toxicity dominating between pH 4.5 to 6, which is an interval identified by McDonald
(1983) that enables Al to bind in the alkaline gill microclimate (pH 6–6.8). However, in these humic streams, under unaltered natural conditions, little Al toxicity was found above pH 5, possibly due to the organic Al complexes that prevailed above this pH. 4.1. Fish response The parallel effect of elevated Ali and lowered pH levels could be observed between the different streams (Figs. 2 and 3) with respect to both the accumulation of Al on the gills and the effect on the blood physiology. The burden of Al on the gills (3–264 μg/g dw) was comparable to previously reported levels for salmon in acidic waters (up to 300 μg/g dw, Monette and McCormick, 2008), but the levels were lower than in simulated mixing zones between acidic and limed water (with transient Al polymers up to 1.5 mg/g dw, Kroglund et al., 2001; Teien et al., 2006). The fish were visibly affected (passive or swimming erratically) by exposure in the Al-rich acidic stream, and affected fish had significantly increased levels of Al on the gills and Hb in the blood. We interpret this as an indication of hypoxia. We suggest that as more Al accumulated on the gills and less oxygen was available to the fish this was compensated by releasing even more Hb to transport oxygen. However, the Hb decreased at the maximum Ali exposure (> 50 μg/L) after a plateau, which suggests that the spleen Hb reservoir was depleted. A similar plateau curve for Hb has been observed for exercised and stressed rainbow trout (Pearson and Stevens, 1991). Significant increases in both gill-Al and Hb were found between the affected and unaffected fish when the results were divided into ranges of
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a) 300
429
c) 150 P-Cl (mmol/L)
Al (g/g dw)
250 200 150 100
100
50
50 N=12-4
0
0 N=1
***
***
26-22 7
18-1 35
37
23
120
<4.6 4.6- 4.8- 5.0- 5.2- 5.4- 5.6- >5.8 4.8 5.0 5.2 5.4 5.6 5.8
<4.6 4.6- 4.8- 5.0- 5.2- 5.4- 5.6- >5.8 4.8 5.0 5.2 5.4 5.6 5.8
pH range
pH range
d)
Glu (mmol/L)
b) 150
Hb (g/L)
100
50
20,0
15,0
10,0
5,0 0 N=1-8
10-3
***
***
,0 N=1-8
12-3
<4.6 4.6- 4.8- 5.0- 5.2- 5.4- 5.6- >5.8 4.8 5.0 5.2 5.4 5.6 5.8
<4.6 4.6- 4.8- 5.0- 5.2- 5.4- 5.6- >5.8 4.8 5.0 5.2 5.6 5.8
pH range
pH range
Fig. 5. Box plots with fish response and condition plotted against water quality (pH split into ranges). Affected fish (black hatched bars) had slow movements, were barely breathing or swam upside down. pH range and a) Al accumulated on the gills, b) Hb, c) P-Cl and d) Glu. Lines in boxes are medians, the ends of the boxes are quartiles, dots represent individual values, and whiskers show the range of values. Outliers (°) are defined as the results outside the quartiles, while extremes (*) are more than three quartiles from the median. Statistically significant differences (t-test) are denoted by *** (P b 0.001) or ** (P b 0.01). For P-Cl, all boxes, and for other variables, the boxes in the ranges with few observations (b6) have their number of samples (N) displayed.
increasing Ali exposure (Fig. 4). The pH effect on the salt balance was not statistically significant, even if the P-Cl generally dropped as anticipated when the water was more acidic (Fig. 5). The effect on P-Cl could not be tested because less blood was available, which might indicate that circulation collapsed at low P-Cl levels. There was a statistically significant effect for variables that reflected the gas exchange linked to the Ali, while pure pH effects could not be confirmed statistically. The lack of plasma (caused by increased blood viscosity) was an indication of the cardiovascular collapse (effects on fluid exchange, hemoconcentration and blood viscosity) proposed in the model for acute acidic toxicity (Milligan and Wood, 1982) and applied to Al toxicity (Dussault et al., 2001). In this experiment, the statistically significant toxic mechanism seemed to be respiratory and (not testable) eventual cardiovascular effects. Similar trend in responses with increased Hb levels accompanied by increased Glu (indicating stress) have been observed in mixing zones with transitory Al chemistry that disturbed the respiratory function (Witters et al., 1996) although at higher Al-gill burden. Therefore many of the responses we observed in Hb, Glu, and higher Al-gill burden here suggest that respiration was the crucial function affected. Al primarily disturbed gas exchange in the gill (oxygen and carbon dioxide), as previously proposed from in vitro studies in less humic waters. The fact that Ali constrains the survival of brown trout is in contrast to what Serrano et al. (2008) proposed in similar TOC-rich waters, namely that high concentrations of TOC and low pH were the main sources of stress and death in the acidic episodes. Also from our study it would not have been possible to elucidate the toxic actions by solely studying the mortality and water
chemistry. Our findings are based on monomeric Ali determinations, coupled with a thorough test of the Ali impact that followed the cause and effect chain through physiological measurements (water– gill–blood) in a large dataset. 4.2. Thresholds Several thresholds can be discerned for the investigated humic streams (Fig. 6). Ali had two thresholds: no toxic effect below 20 μg/L Ali, and physiological effects and some mortality up to 50 μg/L, with high mortality occurring above 50 μg/L. For pH, only one threshold was identified: below pH 5.0, physiological effects and mortality appeared. Comparable bounds for pH (>5.5) and Ali (b33 μg/L) (expressed as monomeric and not as the original total Ali) have been established in a controlled exposure simulating a gradient mixing humic, acidic and Al-rich water with lime (Andrén et al., 2006). Laudon et al. (2005) proposed a model that forecasted brown trout mortality at pH 4.9–5.2 and 15 mg TOC/L finding the ratio of the acid neutralising capacity divided by the hydrogen protons (ANC/H +, selected by a stepwise multiple regressions) to best reflect the fish responses in another in-situ trial in northern Swedish humic spring floods. But, that study gave marginal attention to Ali, which was unfortunate because the toxicities of pH and Al are tightly linked, and Ali can vary locally. In that study, the Ali analysis included polymeric Al forms and not exclusively monomeric Ali; therefore, the conclusion that a high level of Al complexes (correlated to Ali) predisposes brown trout to episodic acidic damage was not surprising. If Al accumulation on the gills had been included in that study together
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Fig. 6. Means values for groups of fish from each sampling occasion (6 living trout/sampling); pH and Ali correlated to accumulation of gill-Al and response in blood parameters (Hb, P-Cl and Glu). Circles represents healthy fish groups (from exposures where no fish have died so far) and crosses fish groups where death has occurred (exposures where fish have died at some time from the stocking).
with monomeric inorganic Ali (Andrén and Rydin, 2009; cf Teien et al., 2005), a more mechanistically sound model would have probably determined that Ali was a more relevant predictor of toxicity. To further elucidate the thresholds, the three-dimensional graph in Fig. 7 shows three discernible groups that confirm the apparent limits from Fig. 6: no effect, physiological effects and mortality. Here, it is even more apparent that to avoid acute toxicity pH should
be above 5.0 and Ali below 20 μg/L which we suggests can be regarded as optimal water quality also for brown trout populations. The group in the middle had little mortality but physiological effects (Ali 20–50 μg/L) and is considered to reflect sub-optimal conditions which the fish can recover from but where growth can be limited. The Ali limit (b20 μg/L) established in our designed in-stream experiment validates the bounds predicted in prior inductive statistical
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Fig. 7. Means values for groups of fish from each sampling occasion (6 living trout/sampling) for accumulated gill Al correlated to pH and Ali. Circles and dashed lines represent healthy fish groups (from exposures where no fish have died so far) and crosses and solid lines are fish groups where death has occurred (exposures where fish have died at some time from the stocking).
studies that correlated water quality and brown trout populations in Norway (11 μg/L, Bulger et al., 1993), the USA (22 μg/L, Baldigo and Lawrence, 2001), Sweden (30 μg/L, Andrén and Bergquist, 2000) and the UK (40 μg/L, Sadler and Turnpenny, 1986). The fact that the thresholds are of similar magnitude confirms the assumption that conditions with no physiological effects during an exposure can be equated with water qualities that can support the residing fish. These thresholds can be used to cautiously decrease liming because both Ali and pH levels have to be balanced to prevent harm to the recovering aquatic biota. Although Ali is tightly linked to pH, the varying Al concentrations in the soil and bedrock as well as local conditions can influence Al release; therefore, it is prudent to monitor Ali. In situ, we have determined the threshold concentrations for Ali and pH for brown trout in moderately acidic humic running waters in Sweden. The pH should be above 5.0 and the Ali should be less than 20 μg/L to sustain brown trout during episodic events. In addition to the effects of low pH we found that Al accumulated which might restrict gill permeability and gas transfer. The thresholds can be used to support liming strategies for recovering fresh waters as the soil locally continues to leak Al, even though acid deposition has diminished after half a century of acid rain. Acknowledgements This work was supported by the Swedish Environmental Protection Agency and the liming programme. The authors thank Paul Andersson (F:a SBV-analys) for making this study possible with his inexhaustible efforts during the intense spring flood field work, and Frode Kroglund (Norwegian Institute for Water Research) for introducing us to fish exposures with physiological sampling as well as discussing the impact of aluminium and acidity. We are most grateful to two anonymous referees who gave valuable comments which improved our paper. References Andrén CM, Bergquist BC. Aluminium and damage to fish populations in limed streams. Acid rain 2000, 6th International Conference on Acidic Deposition, 10–16 December 2000, Tsukuba, Japan. Kluwer Academic Publishers; 2000. p. 321.
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Contents lists available at SciVerse ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Toxicity of inorganic aluminium at spring snowmelt—In-stream bioassays with brown trout (Salmo trutta L.) Cecilia M. Andrén a,⁎, Emil Rydin b a b
Dept. of Applied Environmental Science, ITM, Stockholm University, S-106 91 Stockholm, Sweden Erken Laboratory, Dept. of Ecology and Genetics, Uppsala University, S-761 73 Norrtälje, Sweden
H I G H L I G H T S ► ► ► ► ►
Brown trout were exposed in situ to a gradient of pH and Ali in humic streams. pH above 5.0 and Ali below 20 μg/L sustain healthy brown trout populations. Low pH resulted in a tendency to decreased blood chloride levels. High Ali increased gill Al and haemoglobin suggesting that respiration was effected. The thresholds can be used to support liming strategies in recovering freshwaters.
a r t i c l e
i n f o
Article history: Received 13 March 2012 Received in revised form 31 July 2012 Accepted 1 August 2012 Available online xxxx Keywords: Acidification Brown trout Thresholds Ali & pH Gill accumulation Al Blood physiology Liming strategy
a b s t r a c t Although the acid load has decreased throughout Scandinavia, acidic soils still mobilise aluminium (Al) that is harmful to brown trout. We hypothesise that there are thresholds for Al toxicity and that the toxicity can be traced from the water content to gill accumulation and the consequential physiological effects. During snowmelt, yearlings were exposed to a gradient of pH and inorganic monomeric Al (Ali) in humic streams to study the toxic effects and mortality. Gill Al and physiological blood analyses [haemoglobin (Hb), plasma chloride (P-Cl) and glucose (Glu)] were measured. As the water quality deteriorated, Al accumulated on the gills; Hb and Glu increased; P-Cl decreased, and mortality occurred. Moribund fish had significantly increased gill Al and Hb, suggesting that respiratory disturbances contributed to mortality. Decreased P-Cl and plasma availability indicated an ion regulatory disturbance and possibly circulatory collapse. Ali should be less than 20 μg/L, and pH higher than 5.0, to sustain healthy brown trout populations. These thresholds can be used to fine-tune lime dose, as both Ali and pH levels have to be balanced to prevent harm in the recovering aquatic biota. Although Al is tightly linked to pH, local variation in Al availability in soil and bedrock affects the Al release and subsequent toxic Ali episodes in some catchment areas. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Freshwater bodies in base-poor areas in Europe and North America are recovering chemically from acidification (Skjelkvale et al., 2005; Stoddard et al., 1999) due to decreased sulphur (S) deposition. In areas with deep soil layers, acidification effects might be prolonged as the soil stores S. Many soils still have a large store of organic S, and differences in the rate of decrease between S deposition and runoff cause a considerable time lag, with recovery delayed for decades
⁎ Corresponding author. Tel.: +46 73 707 89 99; fax: +46 8 674 76 36. E-mail addresses: [email protected] (C.M. Andrén), [email protected] (E. Rydin). 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2012.08.006
(Morth et al., 2005; Wright et al., 2005). Furthermore, climate change disturbs runoff patterns and creates hydrological episodes (Arnell, 1999; Bergstrom et al., 2001). These episodes mobilise S and cause acid surges that are harmful to biota (e.g. Kroglund et al., 2008; Lepori and Ormerod, 2005; McCormick et al., 2009). Few records of biological recovery have been documented (Wright et al., 2005), except in Norwegian clear waters (e.g. Raddum et al., 2001) with catchments draining bedrock with shallow soil layers, which may recover faster than areas with deeper soil layers. In North American field studies on brook trout (Salvelinus fontinalis), similar mortality was recorded for 2001–2003 as in previous studies (1984–1985, 1988–1990, and 1997), suggesting no improvement in water quality (Baldigo et al., 2007). Soil aluminium (Al) is dissolved and solubilised in water by low pH, generating inorganic monomeric Al (Ali), which is highly toxic to fish (Driscoll and Schecher, 1990; Gensemer and Playle, 1999). The mere presence of Ali in surface waters has been argued as an
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unambiguous indication of anthropogenic acidification (Lawrence et al., 2007). Fish death in acidic Al-rich water can be caused by several dysfunctions: ion regulatory disturbances (a net loss of plasma electrolytes and a net gain of plasma-H+ and -Al); respiratory dysfunction (concurrent hypoxia, hypercapnia and plasma acidosis); and osmoregulatory breakdown (a net influx of water) (Exley et al., 1991). Al accumulates in the alkaline microenvironment of the gill, leading to tissue damage (Muniz and Leivestad, 1980; Playle and Wood, 1989) that reduces the oxygen and carbon dioxide exchange (Wood and McDonald, 1987) but also disturb ion and osmoregulation. However, pH toxicity has no effect on gas exchange but primarily disturbs ion regulation (inhibits transcellular Na + and Cl − uptake) and osmoregulation (opens paracellular channels) (Wood and McDonald, 1987). Brown trout (Salmo trutta L.) is less vulnerable to acidity than salmon (Salmo salar L.) (Poleo et al., 1997) and is a key goal for lime treatment in Sweden (SEPA, 2010). To plan and balance the lime dose, given that Al availability varies locally, knowledge regarding the environmental effects by concomitant Al-pH toxicity is required. In this study, we expose brown trout to a naturally occurring Ali and pH gradient in humic streams to determine when Ali and pH become toxic to yearling brown trout. We trace the Al effect via accumulation on the gills and the consequential physiological effects. Furthermore, we aimed at distinguishing the Al toxicity from the pH toxicity via the physiological effects.
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2. Materials and methods 2.1. Study area The study was performed in humic streams on coniferous hills located in a base-poor area in the middle of Sweden, where the acid deposition is quite low (Fig. 1). The catchments' buffering capacities are low with granite and gneiss bedrock overlain by a thin layer of till. At springtime in this region, the snow melt flood decreases the pH and increases Ali; although the water's humic content might moderate the Ali toxicity. All streams had minor wetlands upstream of the exposure sites in addition to forest (Table 1). The streams are pristine besides some foresting activities; however, downstream of the Havssvalgsbäcken site, lime was spread before 2000. This lime could have accumulated in the lake 2.5 km upstream the site in Örvallsbäcken and might still influence this stream when strong undercurrents re-suspend and dissolve the lime from the sediment. The streams were chosen based on known water quality to obtain a gradient for the trout exposures: from acidic to slightly acidic to neutral conditions during the snowmelt. All streams have or have had native brown trout populations, with densities ranging from 0 to 77 trout/100 m 2 estimated by electrofishing autumn 2002 (Table 1). In the headwaters (Havssvalgsbäcken), the brown trout population was first diminished by wetland trenching until the fish apparently vanished by acidification and liming began in the early eighties. The
o 4065
Lat. 61°43' Long.16°40' o 4125
o 4250
4041 o
4070 o
o
4081
Fig. 1. Catchment areas and stream exposure sites, with forest (green), wetland (brown), brooks and Lake Örvallssjön (blue). The inserted map shows the location of the study area in Sweden.
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Table 1 Major characteristics of the six stream cage sites. Brooks (site)
Catchment area (km2)
Altitude (m a.s.l.)
Length (km)
Örvallsbäcken (4250) Stängmyrbäcken (4065) Lövåsbäcken (4125) Brånvallsbäcken (4041) Häståbäcken (4081) Havssvalgsbäcken (4070)
6.6 4.8 4.8 4.3 4.7 2.0
114 111 160 153 263 317
6.5 6.7 6.3 3.3 6.9 2.2
a
Land use Forest
Wetland
Water
Other
86% 97% 92% 83% 80% 74%
12% 2% 8% 17% 20% 26%
2% 0% 0% 0% 0% 0%
0% 1% 0% 0% 0% 0%
Populationa trout/100 m2 19 26 77 22 22 0
Brown trout density electro fishing 2002.
mires have now mended and the water quality has improved and a few adult trout have been observed since 2009; though prior to 1940, these were some of the best trout waters in the area according to local fishermen.
2.2. Fish rearing For the exposures, brown trout yearlings (S. trutta L.) (mean 11 cm and 14 g) were used from a nearby hatchery (Bogården fish farm in Ljusnan) also humic but more neutral water quality (pH c.7 and TOC c. 16 mg/L). A local strain, Mellan–Ljusnan, was stocked at all sites at the start of the trials (60 trout/cage) and restocked later in smaller quantities when necessary. This strain was similar to indigenous stream strains and was not expected to have physiological responses caused by differences in water quality at the site of rearing (McWilliams, 1980). Control samples of gills were taken twice (6 + 6 trouts) when the fish were stocked in the cages.
2.3. Fish handling The fish were kept in cages made of stainless steel drums from top loading washing machines, c. 50 L holding 60 trouts i.e. 13.2 kg/m 3. These cages enabled free water passage without allowing the fish to escape and were stable against the stream current. They are commonly used for storing small fish as bait for fishing. The fish cages were checked at each water sampling (see Section 2.5, twice a week and in the acid stream daily). Dead fish were removed, and the cage positions were adjusted to ensure sufficient water flow and avoiding stressful eddies. All fish handling was performed with care according to the study licence (C75/2 2002) from the Uppsala Animal Testing Ethical Council at Tierp District Court.
2.4. Fish sampling Each stream exposure site was visited as often as possible to collect water and fish samples at the same time. The plan was to sample twice a week or more based on apparent fish responses. One stream required more or less daily checks, while the other five were sampled according to the plan. Groups of six trout with normal behaviour were chosen for sampling, but also fish with altered behaviour were collected. For each individual fish, potential altered behaviour was noted, e.g., if they moved slowly, barely breathed or swam upside down, and these moribund fish were designated as affected (as opposed to fit and normal). For comparison, gills from a few dead fishes were also sampled to compare gill-Al with fishes with altered behaviour. Gill-Al data from dead fish is uncertain and should be used carefully. Fish were anesthetised by a blow to the head. Blood samples were taken from the caudal vein after the tail was cut with a sterile scalpel. After obtaining a gill arch sample, the fish were killed by mechanical destruction of the brain.
2.5. Fish analysis Haemoglobin (Hb) and glucose (Glu) were determined on site with Hemocue microcuvettes and spectrophotometers (Hemocue, Ängelholm, Sweden). The remaining blood sample was centrifuged at 2000 ×g for 5 min within 15 min of sampling. The supernatant plasma was removed by pipetting and preserved by freezing; plasma-chloride (P-Cl) was determined with a Radiometer CMT-10 chloride titrator (Radiometer, Copenhagen, Denmark). The second gill arch on the left side was excised from the fish and frozen in a pre-weighed, acid-washed plastic bottle. The gill arch was freeze-dried, weighed and digested in 10% HNO3. The Al in the gill (gill-Al) was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES), and the results were reported as μg/g gill dry weight (dw). 2.6. Water sampling and analysis Water samples were collected twice weekly except in the most acid stream where daily samples were collected at high flow. Samples were taken in polythene bottles that were rinsed with stream water, submersed below the surface and filled to the brim; the cap was screwed on tightly without air in the bottle. Separate bottles were used for the different analyses which were performed with standard analytical methods following quality assurance and control procedures (within the laboratory accreditation from SWEDAC following ISO/IEC 17025). Four variables were used to characterise the water quality. The pH was measured in un-aerated samples with a glass combination electrode (Radiometer). The Al fractions were measured within 1 day as the total monomeric Al (Alm) and organic monomeric Al (Alo) (Driscoll, 1984) by the colorimetric reaction of pyrocatechol violet (Dougan and Wilson, 1974) without acid addition combined with cation exchange using continuous flow analysis with an AutoAnalyzer (Technicon Corp., Tarrytown, New York, USA) at 580 nm. The Alo passed through the column, while the potentially toxic fraction, Ali, was retained by the cation exchange resin (and hence could be calculated as the difference between Alm and Alo). The analytical method for inorganic Al used here exclusively detects monomeric Ali, low molecular mass Al (i.e., Al 3 +, Al(OH)2+, Al(OH) 2 +, AlF2+, AlF 2 +, Al(SO4) +), not including high molecular mass Al polymers (Andrén and Rydin, 2009). The variability for the Ali duplicate samples expressed as relative standard deviation was below 10%, and the detection limit was 3 μg/L. Values below this limit were given the nominal value of 2 μg/L; however, when the Alo exceeded the Alm, the Ali was set to 0 μg/L. The total organic carbon (TOC) was determined by the carbon analyser Shimadzu TOC5000, and calcium was determined with flame atomic absorption spectrometry (AAS). 2.7. Experimental conditions The brown trout were exposed between 21 March and 26 April 2002. In this year, the snowmelt and spring flood were quite moderate
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(discharge 20–670 L/s) after an initial melt when the fish were stocked. The temperature increased in the water from 0 °C to 7.8 °C as spring advanced with lowest temperatures found at the acid site (0.8º ±0.5 °C) which was located at the highest elevation (317 m.a.sl.). Yet a wide spectrum of conditions was covered among the sites (Table 2), with pH values ranging from 4.6 to 6.8 and Ali ranging from 0 to 70 μg/L. There were no confluences between water of different qualities (such as acid and more neutral or limed water) nearby the cage sites so the experiment was conducted in fairly stable waters not affected by mixing zones. Still a test was done at the sites prior to the study that showed marginal difference between field fractionation and lab fractionation within 24 h (unpubl data). The fluctuations in pH and Ali were considered to mainly be caused by snow melting and variances in discharge. The maximum exposure period was 9 days at the acid site and 37 days (the entire melting period) at the other sites. Therefore in order to follow the conditions throughout the snow-melt the acid stream was restocked five times as the trouts then either had died or were spent for sampling. There was no significant separate effect of the exposure time on mortality or any other fish parameter beyond that of the site (two-way ANOVA factors: site and exposure length b9 days or >9 days, P b 0.01).
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3. Results 3.1. Fish response All fish survived in five of the six streams even if their exposure time covered the entire snowmelt period (37 days). In the acid stream though, mortality varied between 10 and 100% for five consecutive rounds of exposure (maximum exposure length 9 days) and was associated with low pH and elevated levels of Ali (Fig. 2). The overall water quality encompassed a wide pH (4.6–6.8) and Ali range (0–69 μg/L) and fluctuated both over time and between sites. The Ali varied with pH (r 2 = 0.80, n = 73, P b 0.001), and when the pH dropped, the Ali increased. The gill-Al in the fish sampled prior to the exposure was (8 ± 7 μg/g dw, n = 12) and represents the background levels in the fish. When the fish were exposed, the accumulated amount of gill-Al (3–264 μg/g dw) and the variation (s.d. 40 μg/g dw) was higher than the background levels. The gill-Al was correlated with the Ali and pH (r 2 = 0.44 and 0.42, respectively, n = 73, P b 0.001). The Hb (66–153 g/L) was also correlated with the Ali and pH (r 2 = 0.47 and 0.57, respectively, n = 62, P b 0.001). The Hb showed high variation (s.d. 16 g/L) with the highest levels (115–153 g/L) found in the fish from the most acidic and Ali-rich stream. Lower Hb levels (66–129 g/L) with less variation (s.d. 10 g/ L) were noted for the fish exposed to more neutral streams. In contrast to Hb the levels of P-Cl (30–110 mmol/L) accompanied pH development; low pH corresponded to lower levels of P-Cl (correlation to Ali, r 2 = 0.59 and pH, r 2 = 0.69, n = 60, P b 0.001). Glu levels (2.2–18.3 mmol/L) were consistently higher at low pH and high Ali (correlation to Ali, r 2 = 0.61 and pH, r 2 = 0.70, n = 62, P b 0.001). The accumulated gill-Al was in turn correlated to increased Hb and Glu and decreased P-Cl (Hb, r 2 = 0.26; Glu, r 2 = 0.36; P-Cl, r 2 = 0.44; n = 62, 62, and 60, respectively, P b 0.001). The effects of stream water conditions on fish response were more apparent when grouped by exposure (n = 6 individuals/sampling) and ordered by decreasing pH (Fig. 3). In the acid stream, fish had more Al accumulated on the gills and showed higher Hb levels. P-Cl decreased and Glu increased in the more acidic water compared to neutral conditions. The repeated rounds of exposure at the acid site showed consistent results. All exposures fell into three categories based on water quality: neutral, slightly acidic and acid water (ANOVA Tukey HSD, P b 0.001) defining the different water qualities of the exposures based on the variables pH and Ali. This division into three categories resulted in nearly equal numbers of observations per category; neutral (18, 24), slightly acidic (24, 28) and acid (18, 21) for blood physiology and gill-Al respectively, thereby enhancing the statistical relevance. For the fish in all three exposure categories, the gill-Al, P-Cl and Glu displayed significant differences (ANOVA Tukey HSD, P b 0.001). For the Hb, only the acidic water was strongly significantly different from the other two categories (P b 0.001) and weakly significantly different (P b 0.05) between the neutral and slightly acidic waters.
2.8. Fish mortality The mortality was calculated in two ways, proportional (%) and categorical (0/+). The proportional mortality was calculated as the share of dead fish out of exposed fish (adjusted for fish removed for sampling) i.e., the total number of dead fish at the site divided by the number of alive fish left at the site. The observed death or accumulated categorical mortality (denoted with crosses in contrast to healthy fish denoted with o), was used for groups of fish (n = 6) and defined as if any mortalities had occurred for this exposure (stocking per stream) since the start of the exposure. 2.9. Statistical analyses The results were divided into different scales to make the study more transparent: individual fish [gill-Al (n = 446), Hb (n = 370), Glu (n = 362), and P-Cl (n = 290)], grouped (six fish from each sampling occasion, n = 73) or separate rounds of exposures (i.e., stockings/stream; 5 streams were stocked once, and there were 5 consecutive stockings in the acid stream, n = 10). Statistics was calculated with SPSS for Windows release 15.0.0 (SPSS Inc., Chicago, Illinois, USA) and presented as average, standard deviation (s.d.) and range (minimum–maximum value). Several statistical tests were performed, including Pearson correlation analysis, analysis of variance (one-way and two-way ANOVA) with the Tukey–Kramer Honestly Significant Difference (HSD) tests and one-sided t-tests was used to describe the relationship between the water and fish measurements. To clearly illustrate the link between water quality and fish health, scatter and box plots were used.
Table 2 Water quality in the six stream cage sites for the exposure periods. Brooks (site) Örvallsbäcken (4250) Stängmyrbäcken (4065) Lövåsbäcken (4125) Brånvallsbäcken (4041) Häståbäcken (4081) Havssvalgsbäcken (4070)
pH
Ali (μg/L)
TOC (mg/L)
Ca (meqv/L)
Median
Min
Max
Median
Min
Max
Median
Min
Max
Median
Min
Max
6.4 6.3 6.3 5.5 5.4 4.8
6.2 6.2 6.0 5.3 5.0 4.6
6.7 6.7 6.8 6.2 6.2 5.0
5 6 3 19 26 49
0 0 0 5 4 18
7 12 17 34 44 69
14 11 13 16 16 18
13 9 10 14 12 15
17 12 14 18 18 19
0.14 0.08 0.10 0.07 0.09 0.06
0.11 0.08 0.08 0.07 0.06 0.04
0.19 0.10 0.21 0.09 0.14 0.06
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Site 4065
4125
4041
4081
4070
pH
Ali (µg/L)
Al (µg/g dw)
Hb (g/l)
P-Cl (mmol/L)
Glu (mmol/l)
Mortality (%)
4250
Date Fig. 2. Temporal variation of the variables observed during the spring flood in 2002 for the six streams. The pH and Ali (water) are at the bottom, followed by the Al accumulation (gills), Hb (blood), P-Cl (blood), Glu (blood), individual fish results, and the cumulative mortality at the top.
3.2. Water quality—fish status To scrutinise the effects on Al accumulation, physiology and fish status (health condition) data were divided into levels according to pH (0.2 intervals) and Ali (10 μg/L intervals) within the pH and Ali ranges (Figs. 4 and 5). Affected moribund fish (swimming erratically and hypo ventilating) had more Al accumulated on their gills compared to unaffected fish within the same Ali level (20–30, 30–40,
40–50 μg Ali/L, t-test, P b 0.001, and > 50 μg Ali/L, P b 0.01, Fig. 4). The Hb increased with the Ali below 50 μg Ali/L, with significantly higher levels for affected fish at the same Ali levels (t-test, P b 0.001); however, above 50 μg Ali/L, the Hb levels dropped. In two pH classes, more Al accumulated in affected fish compared to unaffected fish within the same level (4.6–4.8, 5.0–5.2, t-test, P b 0.001, Fig. 5), which can be expected as Ali is tightly linked to pH. There were too few cases (as affected fish had thickened blood and not
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b)
c)
d)
Hb (g/L)
Ali (µg/L)
a)
427
e)
f)
Fig. 3. Boxplots showing the exposure conditions and fish responses for each site and round of exposure (five consecutive rounds of exposures at the acid site are shadowed in grey); a) pH; b) Ali; c) Al accumulated on the gills; d) Hb; e) P-Cl; and f) Glu. Site numbers are shown below the x-axes and days of exposure above the x-axes. The panels' a–d and f all have data for the same ten boxes. Please note that not enough blood could be sampled to determine the P-Cl in the first exposure at the acid site so only nine boxes are shown in panel e. Lines in boxes are medians; the ends of boxes are quartiles; dots represent individual values, and whiskers show the range of values. Outliers (°) are defined as results outside the quartiles, while extremes (*) are more than three quartiles from the median.
enough plasma for Cl analysis) to statistically test the difference in fish condition for plasma-Cl. There was a trend of lower P-Cl in the affected fish as pH decreased (Fig. 5). Moribund fish with affected behaviour had comparable levels of accumulated Al compared with the dead fish (t-test, P b 0.001). 4. Discussion We showed that Ali occurred in detectable concentrations in humic boreal streams in central Sweden, an unambiguous indication of acidic deposition (Lawrence et al., 2007). Ali was harmful at the slightly acidic sites and deadly to brown trout at the most acidic site. Subsequently, the ongoing influence of suboptimal water qualities might be detrimental to present-day brown trout residents as
noted by the scarcity of trout at the acidic site (Table 1). The analysed Ali consisted only of monomeric forms, which were directly linked to toxic and mortality effects. Moreover, the Ali results showed significant correlations to pH and gill-Al, with no evident amelioration by the relatively high TOC levels (observed by e.g. Neville, 1985; Roy and Campbell, 1997). The strong link to both pH and gill-Al suggests that there was little or no effect of storage time on Ali concentration before analysis opposing previous suggestions in studies with less focus on Al chemistry (Laudon et al., 2005; Serrano et al., 2008). These inorganic monomeric cationic form of Al that can be detected with laboratory fractionation occurs in acidic water (as in this study) unlike the polymeric highly toxic and transient Al complexes found in mixing zones between acidic and limed waters (Poleo, 1995). In this study, we demonstrated toxicity from Ali and pH but
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a)
c)
300
150
P-Cl (mmol/L)
Al (µg/g dw)
250 200 150 100
100
50
50 0 <10
10-20
***
***
***
**
20-30
30-40
40-50
>50
0 N=102 <10
Ali range (µg/L)
63
42-1
38-1
11-10
11-11
*** 10-20
20-30
30-40
40-50
>50
10-20 20-30 30-40 40-50
>50
Ali range (µg/L)
b) 150
d) 20,0
Glu (mmol/L)
15,0
Hb (g/L)
100
50
10,0
5,0
0 <10
10-20
***
***
***
20-30
30-40
40-50
0,0 >50
*** <10
Ali range (µg/L)
Ali range (µg/L)
Fig. 4. Box plots with fish response and condition plotted against water quality (Ali split into ranges). Affected fish (black hatched bars) had slow movements, were barely breathing or swam upside down. Ali range and a) Al accumulated on the gills, b) Hb, c) P-Cl and d) Glu. Lines in boxes are medians, the ends of the boxes are quartiles, dots represent individual values, and whiskers show the range of values. Outliers (°) are defined as results outside the quartiles, while extremes (*) are more than three quartiles from the median. Statistically significant differences (t-test) are denoted by *** (P b 0.001) or ** (P b 0.01). Numbers of samples (N) are displayed for P-Cl, which had few observations in some ranges. All other boxes are based on at least six samples (one fish group sampling).
it was not possible to separate the toxic effects of low pH and high Ali; nonetheless, the results showed that both toxic agents are operative even though the waters were humic. Therefore, given that the need for lime is currently declining, efforts should address the combined effects of Ali and pH and not solely monitor the minimum pH to prevent damage to recovering sensitive trout populations and other biota. Still, only pH, alkalinity, Ca, Mg and colour are routinely measured and Ali is only determined occasionally as the analysis is considered complicated and costly. In our experiments, brown trout mortality was associated with elevated levels of Ali and reduced pH—a link observed for fish in the early days of acidification research (Cronan and Schofield, 1979; Dickson, 1978; Driscoll et al., 1980). The toxicity was, as expected, complex because pH and Ali have parallel and concurrent effects. The Ali level in the water was related to the burden of Al on the gills, as previously documented for other salmonids in streams (Lacroix et al., 1990) as well as in vitro (Playle and Wood, 1989) or by controlled experiments (e.g. Kroglund et al., 2001). The gill-Al in turn affected the physiology related to the Hb, P-Cl and Glu. Separate toxic effects of increased Ali and decreased pH levels could not be differentiated by fish mortality. Combining fish condition with fish physiology through various data clusters, we could partially detect the different effects from pH and Ali. Researchers using salmon (Neville and Campbell, 1988), brown trout fry (Sayer et al., 1991) and brown trout in mixing zones (Witters et al., 1996) have documented concomitant Al and pH effects, with Al toxicity dominating between pH 4.5 to 6, which is an interval identified by McDonald
(1983) that enables Al to bind in the alkaline gill microclimate (pH 6–6.8). However, in these humic streams, under unaltered natural conditions, little Al toxicity was found above pH 5, possibly due to the organic Al complexes that prevailed above this pH. 4.1. Fish response The parallel effect of elevated Ali and lowered pH levels could be observed between the different streams (Figs. 2 and 3) with respect to both the accumulation of Al on the gills and the effect on the blood physiology. The burden of Al on the gills (3–264 μg/g dw) was comparable to previously reported levels for salmon in acidic waters (up to 300 μg/g dw, Monette and McCormick, 2008), but the levels were lower than in simulated mixing zones between acidic and limed water (with transient Al polymers up to 1.5 mg/g dw, Kroglund et al., 2001; Teien et al., 2006). The fish were visibly affected (passive or swimming erratically) by exposure in the Al-rich acidic stream, and affected fish had significantly increased levels of Al on the gills and Hb in the blood. We interpret this as an indication of hypoxia. We suggest that as more Al accumulated on the gills and less oxygen was available to the fish this was compensated by releasing even more Hb to transport oxygen. However, the Hb decreased at the maximum Ali exposure (> 50 μg/L) after a plateau, which suggests that the spleen Hb reservoir was depleted. A similar plateau curve for Hb has been observed for exercised and stressed rainbow trout (Pearson and Stevens, 1991). Significant increases in both gill-Al and Hb were found between the affected and unaffected fish when the results were divided into ranges of
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a) 300
429
c) 150 P-Cl (mmol/L)
Al (g/g dw)
250 200 150 100
100
50
50 N=12-4
0
0 N=1
***
***
26-22 7
18-1 35
37
23
120
<4.6 4.6- 4.8- 5.0- 5.2- 5.4- 5.6- >5.8 4.8 5.0 5.2 5.4 5.6 5.8
<4.6 4.6- 4.8- 5.0- 5.2- 5.4- 5.6- >5.8 4.8 5.0 5.2 5.4 5.6 5.8
pH range
pH range
d)
Glu (mmol/L)
b) 150
Hb (g/L)
100
50
20,0
15,0
10,0
5,0 0 N=1-8
10-3
***
***
,0 N=1-8
12-3
<4.6 4.6- 4.8- 5.0- 5.2- 5.4- 5.6- >5.8 4.8 5.0 5.2 5.4 5.6 5.8
<4.6 4.6- 4.8- 5.0- 5.2- 5.4- 5.6- >5.8 4.8 5.0 5.2 5.6 5.8
pH range
pH range
Fig. 5. Box plots with fish response and condition plotted against water quality (pH split into ranges). Affected fish (black hatched bars) had slow movements, were barely breathing or swam upside down. pH range and a) Al accumulated on the gills, b) Hb, c) P-Cl and d) Glu. Lines in boxes are medians, the ends of the boxes are quartiles, dots represent individual values, and whiskers show the range of values. Outliers (°) are defined as the results outside the quartiles, while extremes (*) are more than three quartiles from the median. Statistically significant differences (t-test) are denoted by *** (P b 0.001) or ** (P b 0.01). For P-Cl, all boxes, and for other variables, the boxes in the ranges with few observations (b6) have their number of samples (N) displayed.
increasing Ali exposure (Fig. 4). The pH effect on the salt balance was not statistically significant, even if the P-Cl generally dropped as anticipated when the water was more acidic (Fig. 5). The effect on P-Cl could not be tested because less blood was available, which might indicate that circulation collapsed at low P-Cl levels. There was a statistically significant effect for variables that reflected the gas exchange linked to the Ali, while pure pH effects could not be confirmed statistically. The lack of plasma (caused by increased blood viscosity) was an indication of the cardiovascular collapse (effects on fluid exchange, hemoconcentration and blood viscosity) proposed in the model for acute acidic toxicity (Milligan and Wood, 1982) and applied to Al toxicity (Dussault et al., 2001). In this experiment, the statistically significant toxic mechanism seemed to be respiratory and (not testable) eventual cardiovascular effects. Similar trend in responses with increased Hb levels accompanied by increased Glu (indicating stress) have been observed in mixing zones with transitory Al chemistry that disturbed the respiratory function (Witters et al., 1996) although at higher Al-gill burden. Therefore many of the responses we observed in Hb, Glu, and higher Al-gill burden here suggest that respiration was the crucial function affected. Al primarily disturbed gas exchange in the gill (oxygen and carbon dioxide), as previously proposed from in vitro studies in less humic waters. The fact that Ali constrains the survival of brown trout is in contrast to what Serrano et al. (2008) proposed in similar TOC-rich waters, namely that high concentrations of TOC and low pH were the main sources of stress and death in the acidic episodes. Also from our study it would not have been possible to elucidate the toxic actions by solely studying the mortality and water
chemistry. Our findings are based on monomeric Ali determinations, coupled with a thorough test of the Ali impact that followed the cause and effect chain through physiological measurements (water– gill–blood) in a large dataset. 4.2. Thresholds Several thresholds can be discerned for the investigated humic streams (Fig. 6). Ali had two thresholds: no toxic effect below 20 μg/L Ali, and physiological effects and some mortality up to 50 μg/L, with high mortality occurring above 50 μg/L. For pH, only one threshold was identified: below pH 5.0, physiological effects and mortality appeared. Comparable bounds for pH (>5.5) and Ali (b33 μg/L) (expressed as monomeric and not as the original total Ali) have been established in a controlled exposure simulating a gradient mixing humic, acidic and Al-rich water with lime (Andrén et al., 2006). Laudon et al. (2005) proposed a model that forecasted brown trout mortality at pH 4.9–5.2 and 15 mg TOC/L finding the ratio of the acid neutralising capacity divided by the hydrogen protons (ANC/H +, selected by a stepwise multiple regressions) to best reflect the fish responses in another in-situ trial in northern Swedish humic spring floods. But, that study gave marginal attention to Ali, which was unfortunate because the toxicities of pH and Al are tightly linked, and Ali can vary locally. In that study, the Ali analysis included polymeric Al forms and not exclusively monomeric Ali; therefore, the conclusion that a high level of Al complexes (correlated to Ali) predisposes brown trout to episodic acidic damage was not surprising. If Al accumulation on the gills had been included in that study together
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Fig. 6. Means values for groups of fish from each sampling occasion (6 living trout/sampling); pH and Ali correlated to accumulation of gill-Al and response in blood parameters (Hb, P-Cl and Glu). Circles represents healthy fish groups (from exposures where no fish have died so far) and crosses fish groups where death has occurred (exposures where fish have died at some time from the stocking).
with monomeric inorganic Ali (Andrén and Rydin, 2009; cf Teien et al., 2005), a more mechanistically sound model would have probably determined that Ali was a more relevant predictor of toxicity. To further elucidate the thresholds, the three-dimensional graph in Fig. 7 shows three discernible groups that confirm the apparent limits from Fig. 6: no effect, physiological effects and mortality. Here, it is even more apparent that to avoid acute toxicity pH should
be above 5.0 and Ali below 20 μg/L which we suggests can be regarded as optimal water quality also for brown trout populations. The group in the middle had little mortality but physiological effects (Ali 20–50 μg/L) and is considered to reflect sub-optimal conditions which the fish can recover from but where growth can be limited. The Ali limit (b20 μg/L) established in our designed in-stream experiment validates the bounds predicted in prior inductive statistical
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Fig. 7. Means values for groups of fish from each sampling occasion (6 living trout/sampling) for accumulated gill Al correlated to pH and Ali. Circles and dashed lines represent healthy fish groups (from exposures where no fish have died so far) and crosses and solid lines are fish groups where death has occurred (exposures where fish have died at some time from the stocking).
studies that correlated water quality and brown trout populations in Norway (11 μg/L, Bulger et al., 1993), the USA (22 μg/L, Baldigo and Lawrence, 2001), Sweden (30 μg/L, Andrén and Bergquist, 2000) and the UK (40 μg/L, Sadler and Turnpenny, 1986). The fact that the thresholds are of similar magnitude confirms the assumption that conditions with no physiological effects during an exposure can be equated with water qualities that can support the residing fish. These thresholds can be used to cautiously decrease liming because both Ali and pH levels have to be balanced to prevent harm to the recovering aquatic biota. Although Ali is tightly linked to pH, the varying Al concentrations in the soil and bedrock as well as local conditions can influence Al release; therefore, it is prudent to monitor Ali. In situ, we have determined the threshold concentrations for Ali and pH for brown trout in moderately acidic humic running waters in Sweden. The pH should be above 5.0 and the Ali should be less than 20 μg/L to sustain brown trout during episodic events. In addition to the effects of low pH we found that Al accumulated which might restrict gill permeability and gas transfer. The thresholds can be used to support liming strategies for recovering fresh waters as the soil locally continues to leak Al, even though acid deposition has diminished after half a century of acid rain. Acknowledgements This work was supported by the Swedish Environmental Protection Agency and the liming programme. The authors thank Paul Andersson (F:a SBV-analys) for making this study possible with his inexhaustible efforts during the intense spring flood field work, and Frode Kroglund (Norwegian Institute for Water Research) for introducing us to fish exposures with physiological sampling as well as discussing the impact of aluminium and acidity. We are most grateful to two anonymous referees who gave valuable comments which improved our paper. References Andrén CM, Bergquist BC. Aluminium and damage to fish populations in limed streams. Acid rain 2000, 6th International Conference on Acidic Deposition, 10–16 December 2000, Tsukuba, Japan. Kluwer Academic Publishers; 2000. p. 321.
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