Reproduction, blood and plasma parameters and gill histology of vendace (Coregonus albula L.) in long-term exposure to acidity and aluminum

Ecotoxicology and Environmental Safety 54 (2003) 255–276

Reproduction, blood and plasma parameters and gill histology of vendace (Coregonus albula L.) in long-term exposure to acidity and aluminum Pekka J. Vuorinen,* Marja Kein.anen, Seppo Peuranen,1 and Christina Tigerstedt Finnish Game and Fisheries Research Institute, P.O. Box 6, FIN-00721 Helsinki, Finland Received 30 May 2001; received in revised form 27 June 2002; accepted 8 July 2002

Abstract Vendace were exposed to pHs 4.75 and 5.25 with or without added aluminum (200 mg=7.4 mmol Al L
1) from late endogenous vitellogenesis in July through the spawning period. At the normal time of spawning, when 48% of the control females had already released their eggs, 50% of females at pH 4.75+Al had completely unovulated oocytes. The final proportions of completely ovulated females were 14%, 36%, 25%, 61%, and 81% at pH 4.75+Al, pH 4.75, pH 5.25+Al, pH 5.25, and in the control group, respectively. Delayed testes regression was seen in males at pH 4.75+Al. A clear decrease in plasma Na+ and Cl
and an increase in blood glucose concentration was detected only near spawning time, from October to November, coincident with Al accumulation inside the gill tissue. It is concluded that seasonal changes, probably related to reproductive physiology or to the decrease in water temperature, are associated with the increase in Al toxicity in vendace. r 2002 Elsevier Science (USA). All rights reserved. Keywords: Vendace; Coregonus; Reproduction; Ovulation; Plasma chloride; Blood glucose; Gill histology; pH; Acidity; Aluminum

1. Introduction In acidified waters, fish stocks have deteriorated and the age distribution has shifted toward a predominance of older age groups (Rask and Tuunainen, 1990). Since the early life stages of fish are in many cases the most sensitive to adverse environmental conditions and toxicants, it has been proposed that fish stocks have declined in acidified waters due to an increased mortality of embryos and fry (Mount et al., 1988b). In addition, acidification might directly affect the reproductive physiology of fish. Beamish et al. (1975) reported that several fish species in George Lake in the La Cloche Mountain lakes district had not released their eggs when examined after their normal spawning time. This was hypothesized to be due to abnormal Ca2+ metabolism, because the female-to-male plasma Ca2+ ratio was low *Corresponding author. Fax: +358-205-751201. E-mail addresses: pekka.vuorinen@rktl.fi (P.J. Vuorinen), marja. keinanen@rktl.fi (M. Kein.anen), [email protected] (S. Peuranen), christina.tigerstedt@rktl.fi (C. Tigerstedt). 1 Present address: Danisco-Cultor Innovation Kantvik, Sokeritehtaantie 20, FIN-02460 Kantvik, Finland.

in these cases. However, this hypothesis was based on quite a small number of males. During normal vitellogenesis, the plasma Ca2+ concentration increases when vitellogenin is transported to the ovaries as a Ca2+-bound complex (Mommsen and Walsh, 1988). Beamish et al. (1975) sampled fish only once and it is possible that spawning was not completely inhibited but was at least partly postponed. This was demonstrated in Finland, where the spawning of perch (Perca fluviatilis) was delayed in two highly acidified lakes (Rask et al., 1990; Vuorinen et al., 1992). Moreover, in whitefish (Coregonus lavaretus wartmanni) exposed for 4 months prior to spawning to low pH and aluminum (Al), a disturbance in the maturation of oocytes, delayed spawning, and delayed after-spawning regression of testes were observed (Vuorinen et al., 1990; Vuorinen and Vuorinen, 1991), and when examined 3 weeks after their normal spawning time, 28% of whitefish exposed to pH 4.75 with added Al had not ovulated (Vuorinen et al., 1990). Ovulation in brook trout (Salvelinus fontinalis) was also significantly delayed in a 10-month exposure to pH 4.5 and 5.2 without added Al (Tam and Payson, 1986) and somewhat when exposed for over 6

0147-6513/03/$ - see front matter r 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S0147-6513(02)00078-7

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months to pH 5.0 with or without added Al (170 mg L
1) in water with low (0.01 mmol L
1) and moderate (0.19 mmol L
1) calcium levels, although they produced mature eggs (Mount et al., 1988a). Delayed ovulation in brook trout and whitefish was related to acidity and the dissolved Al concentration (Mount et al., 1988a; Vuorinen et al., 1990; Vuorinen and Vuorinen, 1991). When whitefish were sampled after monitoring their ovulation rate and readiness to spawn for over 2 weeks, their ion regulation was found to be disturbed at pH 4.75 with or without added Al (150 mg=5.6 mmol L
1) in the exposures in which their reproduction was also affected (Vuorinen et al., 1990). In addition to decreased plasma Na+ and Cl
concentrations, these whitefish also exhibited an accumulation of Al inside the gill tissue and general physiological stress, indicated by an increased blood glucose concentration (Vuorinen et al., 1990; Vuorinen and Vuorinen, 1991). This physiological stress was believed to be behind the delayed egg maturation of the whitefish. The present study on the vendace (Coregonus albula) was designed to complement previous experiments on the whitefish (Vuorinen et al., 1990; Vuorinen and Vuorinen, 1991), a closely related species. Due to their smaller size, vendace could be exposed in greater numbers than whitefish and sampled several times during the exposure period. The vendace is the most important commercial species in inland fisheries in Finland. Although its fisheries are concentrated in large lakes, it is also found in smaller lakes where it is of local importance. Vendace were exposed under laboratory conditions to two acidity levels with or without added Al from late endogenous vitellogenesis throughout the spawning time. The goal of the present study was to investigate the long-term effects of acidity and Al on the timing of spawning of vendace and, more extensively, to histologically examine the development and maturation rates of gametes. In addition to monitoring growth and mortality, we investigated the accumulation of Al in the gill epithelium, gill structure and function, and blood and plasma parameters indicating disturbances in ion balance and oxygen transfer capacity as well as general physiological stress, effects which were earlier demonstrated in whitefish (Vuorinen et al., 1990; Vuorinen and Vuorinen, 1991).

2. Materials and methods 2.1. Fish The experimental fish, about 1500 vendace (Coregonus albula L.), were caught in March by seine net in Lake Pyh.aj.arvi, Finnish Karelia, and transported to the nearby Saimaa Fisheries Research and Aquaculture

Station of the Finnish Game and Fisheries Research Institute. The fish were put into a large indoor concrete basin, 6 m in diameter, where, over a period of 3 months, they became used to taking dry feed (Ewos) and acclimated to the experimental water (from Lake Yl.aEnonvesi). At the start of the experiment, the body weight of vendace was 35.071.9 g (mean7SE, N ¼ 30), the total length 18.770.4 cm, and the condition factor 0.5270.01. Condition factors (CF) were calculated by the formula CF=100  (weight, g)  (length, cm)
3. The mean (7SE) age of the vendace, determined from all vendace sampled in September and October, was 2.170.1 years. 2.2. Experiment Two weeks before the start of the experiment, about 1000 healthy vendace in good condition were randomly distributed between five experimental basins. The basins were of dark green fiberglass with a diameter of 2.7 m and a water volume of about 2000 L. The treatments were as follows: pH 4.75, pH 4.75+Al 200 mg (=7.4 mmol) L
1 (pH 4.75+Al), pH 5.25, pH 5.25+Al 200 mg L
1 (pH 5.25+Al), and a control. The fish were fed with dry feed at 2% of live weight per day. The exposure lasted 145 days, from 6 July to 28 November, finishing about 3 weeks after the spawning time of vendace. The experiment was a flow-through test with a water supply of 0.4 l s
1 and a 90% replacement time of 3 h (see Vuorinen et al., 1990). Water from Lake Yl.aEnonvesi was led into the basins through mixing chambers. The lowering of pH was achieved by pumping acidic stock solutions with peristaltic pumps (Desaga 132100) into mixing chambers, the water of which was aerated vigorously with compressed oil-free air to expel carbon dioxide. The stock solutions were made once a week by adding sulfuric acid (Merck No. 731) into lake water or first dissolving Al2(SO4)3  (16– 18)H2O (Merck No. 1100). Water pH in the test basins was continuously monitored with pH meters (Schott CG817T, electrode N37) connected to recorders (Goertz SE-120). Separate pH values, 4 h apart, were recorded from the charts, and the mean pH values were calculated from these readings after converting them to hydrogen ion concentrations (Table 1). Water temperature varied with that of lake water and was at its maximum (21 C) in early August and then decreased to 2 C toward the end of the experiment. The photoperiod was natural, and one-half of the surface of the basins was covered with black plastic. The dissolved oxygen (DO) content in the water of the test basins was monitored with a DO meter (Yellow Springs Instrument Model No. 57). The percentage saturation of DO was mostly 85–95%, but was at its lowest, approximately 70%, during the warmest water temperature period. For

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257

Table 1 The measured pH values and aluminum speciation in the test waters

pH Total Al (mg L
1) Fast-reactive Al (mg L
1) Slow-reactive Al (mg L
1)

Control

pH 5.25

pH 5.25+Al

pH 4.75

pH 4.75+Al

7.0070.01 (137) 671 — 672

5.2470.00 (143) 1472 571 1371

5.2470.00 (145) 16876 6178 16373

4.7370.01 (142) 1572 — 1471

4.7170.01 (143) 213719 70715 197712

Note: Mean (7SE) pH values are from daily means of the continuous pH recordings, the numbers of which are given in parentheses. Aluminum concentrations (mean7SE) are from the three measurements in September (after 66 days of exposure), October (108 days), and November (145 days).

other water analyses, samples from the five basins were taken in connection with fish samples, three times altogether, and analyzed according to Finnish water analysis standards (SFS standards), apart from fluoride analysis by ion selective electrode (Orion Research). Determination of Al speciation in the test waters was performed at Helsinki University of Technology according to LaZerte (1984) by graphite furnace atomic absorption spectrometry (Table 1) and the remaining metals by atomic absorption in the laboratory of the Uusimaa Regional Environment Centre. In addition, Al speciation was estimated theoretically using the MINEQL+ version 4.0 software (Schecher and McAvoy, 1998), and the speciation of Al was calculated for several water temperatures between 5 C and 25 C. In the dilution lake water the concentrations of heavy metals, except total iron (Fe) (71–75 mg L
1), were below the detection limits; water pH was 7.05–7.13, color 35 mg Pt L
1, conductivity 7.4–7.5 mS m
1, Ca2+ 0.23 mmol L
1 and F
3.5 mmol L
1. 2.3. Sampling and determinations Vendace were sampled three times: 9
11 September (i.e., when exposed for 65
67 days), 21
23 October (on days 107
109), and 24
28 November (on days 141
145), about 3 weeks after the spawning period. In November, only males were sampled, because all the fish identified as matured females had been killed, weighed, and measured at the stripping of eggs (3–5 November; on days 120
122). However, at the final sampling, when all the remaining fish were killed and examined, a few females were found, and their weight and length were recorded. In sampling, fish were randomly caught with a dip net successively from the five basins and individually anesthetized for 3 min in MS-222 solution (tricaine methane sulfonate, 0.13 g L
1) buffered with NaHCO3 to the pH of the appropriate test solution. Blood was sampled by lateral puncture of dorsal blood vessels and drawn into a heparinized 1-mL syringe (needle no. 16; 0.6  25 mm). After blood sampling, each fish was measured and weighed and its liver and gonads were also weighed to calculate proportional weights (LSI and GSI, respec-

tively). At the September sampling of females and at each sampling of males, a sample of gonad from the middle part of the organ was fixed in Bouin’s solution. The developmental stages of oocytes were then recorded in Masson–Gomori-stained paraffin sections of ovaria according to the classification of Zawisza and Backiel (1970). Spermiogenesis and regression of testes were examined in hematoxylin- and eosin-stained paraffin sections. The extent of spermiogenesis in September and October was recorded in arbitrary units ranging from 1 (=no spermatids) to 6 (=numerous spermatids) and, for testes regression after spawning in November, from –8 (=no regression, numerous spermatids) to 12 (=advanced regression, no spermatids remaining). When the spawning time approached, vendace were monitored for spawning behavior. When this behavior was obvious among vendace in the control basin, stripping of eggs from females was started. This was performed between 3 and 5 November. Vendace were caught with a dip net; the females were anesthetized as during sampling and stripped of mature, ovulated eggs. Mature eggs were transparent and easily separated and were extruded by pressing gently on the sides of the fish; by contrast, immature eggs were opaque and stuck together. Any males caught were released into 70-L plastic buckets with appropriately aerated test solution and kept there until stripping was complete. The males were therefore handled only once during stripping. The mass of the stripped eggs was added to the mass of the ovulated eggs that remained in the coelom of the female to obtain the total weight of ovulated eggs. The weights of both ovaries and the liver were also recorded. A sample of unfertilized eggs was taken for determination of fresh and dry weight as well as water content. The proportion of unovulated eggs was estimated from the total egg amount, and the ovulatory state of females was classified according to the degree of ovulation and the proportional weights of the gonads and stripped eggs. The scale was as follows: 1 (=completely unovulated), 2–5 (=increasing degree of ovulation), 6 (=completely ovulated), and 7 (=spent, i.e., eggs already released). Immediately after the blood sample was drawn, subsamples were centrifuged for the hematocrit reading (Hct) (Heraeus Haemofuge A; 12,000 rpm for 3 min)

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and pipetted for the determinations of hemoglobin (Hb), glucose, and lactate concentrations. The rest of the blood was centrifuged (Beckman Microfuge B; 11,600 rpm for 1.5 min) twice to separate blood cells from plasma. Determinations from plasma [calcium ([Ca2+]), magnesium ([Mg2+]), sodium ([Na+]), and chloride ([Cl
]) concentration] and blood, and calculation of the mean corpuscular hemoglobin concentration (MCHC), were performed as described in Vuorinen et al. (1990). The second and third gill arch from the left side of each male vendace were excised, fixed in 4% buffered formaldehyde solution, embedded in paraffin, and processed for histological examination [morphology, morphometric measurements, and counting of mucous cells and aluminum inclusions as described in Vuorinen et al. (1990) and Peuranen et al. (1993, 1994)]. The experiment described complies with the current laws on animal experimentation in Finland (Finnish law 247/96 and act 395/96).

3. Statistics One-way ANOVA was applied to test the effect of the treatments, and the differences between means were tested successively by Tukey’s test at the 95% level. The effects of pH and aluminum were tested by two-way ANOVA, and the dependence of CFs of dead vendace on exposure time was tested by regression analysis. Where there were large differences between the variances, the statistical tests were performed after the variable values had been transformed to ranks. An arcsine transformation was performed for GSI, LSI, and the mucous cell and aluminum deposit numbers before the analysis of variance. The Statistical Analysis System (version 6.12) software program was used to perform the statistical calculations (SAS Institute Inc., 1988).

4. Results 4.1. Aluminum speciation According to the chemical measurements, the labile (fast-reactive) Al concentration was only somewhat higher at pH 4.75 than at pH 5.25 (Table 1). However, the theoretical calculations showed that the proportions of free Al ion and fluoride complexes were greater at the lower than at the higher pH levels, where the proportion of organic complexes was greater (Fig. 1). According to the calculations performed in MINEQL+, the proportions of the Al species in relation to the pH remained similar throughout the range of temperature variation during the test.

Fig. 1. Distribution of Al species calculated with the MINEQL+ software from the nominal aluminum concentration (200 mg=7.4 mmol L
1) at 5 C in the test water (characteristics given under Section 2). Ionic strength correction was applied and the system was considered to be open to the atmosphere.

4.2. Growth and mortality Male vendace sampled in September were significantly smaller in weight and length in pH 4.75+Al than the controls and in October smaller in weight in pH 5.25 (Table 2). There were no other statistically significant differences between the groups at the sampling times in the length or weight of males or females (Tables 2 and 3). On the basis of condition factors, females in October in pH 4.75 and pH 4.75+Al groups were leaner than the controls (Table 3). According to two-way ANOVA, acidity reduced the CF of females in October (Po0:001). Moreover, acidity and Al jointly decreased the length of males in September and acidity and Al significantly reduced the CF values of males both separately (Po0:01) and jointly (Po0:05) in November. Vendace in pH 4.75+Al were seemingly less willing to take feed than in the control and other treatments. The swimming activity of these fish was also reduced; occasionally they were just drifting with the current. The feed intake was also lowered in pH 4.75 and pH 5.25+Al. The CFs of the fish that died during the experiment became lower toward the end of the exposure in pH 5.25+Al, pH 4.75 and pH 4.75+Al; in the last of these the decrease was significant (CF=0.478–0.001days, r2 ¼ 0:17; Po0:001). However, there were no significant differences between the groups in the mean weights, lengths, or CFs of all dead vendace (Table 4). On the other hand, the mean weight and CF of dead vendace in the control and all the exposure groups were significantly (Po0:05) lower than those of the sampled vendace in the respective groups. The numbers of dead females and males was greatest in pH 4.75+Al followed by pH 5.25+Al and pH 4.75 (Table 4). In the exposure groups, except in pH

P.J. Vuorinen et al. / Ecotoxicology and Environmental Safety 54 (2003) 255–276 Table 2 The size of male vendace in treatment groups Month

N

259

Table 4 Mortalities and the sizea of dead vendace in treatment groups

Length (mm)

Weight (g)

CF

N

Mortality (%)

Length (mm)

Weight (g)

CF

September Control pH 5.25 pH 5.25+Al pH 4.75 pH 4.75+Al

6 8 4 7 5

20.070.3a 19.170.3ab 20.070.2ab 19.470.3ab 18.870.5b

50.673.8a 39.972.4ab 41.876.6ab 43.770.9ab 36.271.7b

0.6270.03a 0.5670.02a 0.5270.08a 0.5970.03a 0.5670.03a

Females Control pH 5.25 pH 5.25+Al pH 4.75 pH 4.75+Al

122 130 123 130 126

32.0 31.5 54.4 40.0 53.2

18.670.4 18.770.2 18.070.3 18.670.2 18.270.2

25.471.6 26.171.8 25.171.2 25.771.3 24.271.1

0.4070.02 0.3870.02 0.4270.01 0.3970.02 0.3970.01

October Control pH 5.25 pH 5.25+Al pH 4.75 pH 4.75+Al

6 5 7 6 6

19.170.5a 16.671.0a 18.770.2a 18.170.8a 18.370.9a

41.574.2a 24.272.5b 32.771.7ab 33.173.6ab 32.875.5ab

0.5870.03a 0.5270.04a 0.5070.02a 0.5570.03a 0.5070.02a

Males Control pH 5.25 pH 5.25+Al pH 4.75 pH 4.75+Al

95 81 73 87 88

30.5 32.1 45.2 43.7 68.2

17.770.4 17.470.4 17.070.3 17.670.3 17.170.3

25.771.6 26.671.9 20.971.3 24.871.6 21.071.4

0.4670.02 0.4970.02 0.4170.02 0.4470.02 0.4070.02

November Control pH 5.25 pH 5.25+Al pH 4.75 pH 4.75+Al

34 17 12 17 10

17.070.3a 17.170.4a 17.070.5a 17.170.4a 17.070.7a

26.671.2a 28.871.7a 24.171.9a 24.772.0a 24.872.9a

0.5370.01ab 0.5670.01a 0.4870.02b 0.4870.01b 0.4770.02b

Note: Mortality percentages are based on the original numbers of exposed females and males (N). a The mean (7SE) length, weight, and condition factor (CF) of those female and male vendace that died during the 145-day exposure.

Note: Fish were sampled 9–11 September, 21–23 October (before spawning time), and 24–28 November (about 3 weeks after normal spawning time). N is the number of sampled fish. The mean (7SE) length, weight, and condition factor (CF) are given. The dissimilar letters as a superscript to the SEs indicates that the mean values of the treatment groups within each sampling time differed significantly (Po0:05; Tukey’s test).

5.25+Al, more males than females died during the experiment (calculated as a percentage of exposed males and females, respectively), and deaths occurred at quite a steady rate, except in pH 4.75+Al, where mortality increased sharply after spawning time at the end of the exposure, when water temperature also decreased within a few days from approximately 5 C to 2 C. Mortality was similar in the control and pH 5.25. In these two groups and in pH 5.25+Al the highest death rates occurred during the period of warm summer temperatures, although in pH 5.25+Al the mortality rate toward the end of the exposure was higher than in pH 5.25.

Table 3 The size of female vendace in treatment groups Month

N

Length (mm)

Weight (g)

CF

September Control pH 5.25 pH 5.25+Al pH 4.75 pH 4.75+Al

16 8 10 10 12

19.370.5a 19.670.2a 19.170.2a 19.370.5a 19.470.3a

45.173.6a 45.472.6a 37.772.5a 41.572.9a 41.073.0a

0.5970.02a 0.5870.02a 0.5270.02a 0.5670.02a 0.5470.02a

October Control pH 5.25 pH 5.25+Al pH 4.75 pH 4.75+Al

10 10 7 10 11

18.970.7a 18.670.8a 20.270.5a 19.070.2a 19.170.4a

49.875.1a 48.076.1a 55.478.1a 40.572.2a 39.072.5a

0.5670.02a 0.6270.03ac 0.5470.04ab 0.5270.02bc 0.4970.02b

November Control pH 5.25 pH 5.25+Al pH 4.75 pH 4.75+Al

5 7 3 3 —

19.370.4a 18.670.6a 19.270.6a 19.371.2a —

36.472.9a 34.673.1a 29.473.2a 30.671.1a —

0.5070.02a 0.5370.03a 0.4070.02a 0.4370.06a —

Note: Fish were sampled 9–11 September, 21–23 October (before spawning time), and 24–28 November (about 3 weeks after normal spawning time). N is the number of sampled fish. The mean (7SE) length, weight, and condition factor (CF) are given. The dissimilar letters as a superscript to the SEs indicates that the mean values of the treatment groups within each sampling time differed significantly (Po0:05; Tukey’s test).

4.3. Reproduction The gonadosomatic indices (GSI) of females were less than 5% in September and did not differ significantly between the groups, but the lowest mean GSIs were found in the most severe exposures (Fig. 2). The pattern of oocyte development was most uniform in the control group, although there were no significant differences between groups in the proportions of oocyte developmental stages or of atretic ones (P40:05; Fig. 3). In October, by which time the GSI in females had become 5
7 times greater than in September (Po0:05; Fig. 2), the GSI of control females was highest (21.871.0%) and differed significantly from all the other groups except for pH 5.25+Al (Fig. 2). In September, the GSI of males tended to be lowest in the Al groups but there was considerable variation in the values (Fig. 2). Spermatogenesis was nevertheless most advanced in the control followed by Al groups and was at its earliest stages in the groups exposed to low pH alone (Figs. 4 and 5). The GSI of control males decreased from

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Fig. 2. The mean (7SE) gonadosomatic indices (GSI) of female and male vendace exposed to pH 4.75 and 5.25 with or without added aluminum (200 mg=7.4 mmol L
1) from late endogenous vitellogenesis through the normal spawning period. The figures in November are from the residual weight of ovaries of females after stripping of eggs and in males from sampling 3 weeks after the normal spawning period. The number of fish sampled is in parentheses. Dissimilar letters above the columns indicate significant (Po0:05; Tukey’s test) differences between the groups within each sampling time.

September to October (Fig. 2), and it appeared that in some control fish, sperm had already been partly released; spermiogenesis at this time was less advanced in all the exposure groups than in the control (Figs. 4 and 5). In November, at spawning time, the GSI of females (eggs removed) had declined to the level recorded in September in other groups, except for pH 4.75+Al (Fig. 2). The GSI of the control males continued to decrease from October until November, 3 weeks after spawning time, when the GSI in the pH 4.75+Al group was significantly (Po0:05) higher than in the control group (Fig. 2). Most males in this group and some at pH 4.75 still delivered milt if gently pressed

laterally near the anus. The testes of the fish in both pH 4.75 groups still contained a large number of spermatozoa, while those in the pH 5.25 groups contained moderate or only small amounts; in the testes of control fish hardly any spermatozoa remained (Figs. 4 and 5). According to two-way ANOVA, the treatments had no significant effect on the GSI of females in September, but acidity significantly (Po0:01) decreased the GSI in October and significantly (Po0:001) increased it in November. Aluminum significantly (Po0:05) decreased the GSI of males in September and acidity significantly (Po0:01) increased it in November. The liver somatic indices (LSI) of females were higher in October than in September, although the difference was not significant in the control group. There were no significant differences between the groups at either time, but in September the LSI of females in the Al-exposed groups tended to be lowest, and in October it tended to be somewhat higher in all exposure groups than in the control. Similarly, in males the LSIs of Al-exposed groups tended to be lowest in September, but in October there were no clear trends. In males, LSI did not increase as markedly from September to October as in females, and in males the LSIs were lower at both times than in females. By spawning time, the LSI values had again decreased in females in all groups, and the lowest value (although not significantly different) was in pH 4.75+Al. In September and November, Al significantly (Po0:05) decreased the LSI of males and in October Al and acidity in combination significantly (Po0:05) increased it. Spawning behavior was first observed in the control group and at pH 5.25 without Al on 26 October, and 9 days later, on 4 November, in both pH 4.75 groups. At the normal spawning time, around 3–5 November, when the spawning-readiness of females was examined, 48% of the control females had already released most or all of their eggs, compared with 17% in pH 5.25 and pH 5.25+Al, 9% in pH 4.75, and none in pH 4.75+Al (Fig. 6). The proportions of completely ovulated females were 81% in the control, 61% in pH 5.25, 25% in pH 5.25+Al, 36% in pH 4.75, and 14% in pH 4.75+Al. In half of the females in pH 4.75+Al the eggs were not ovulated at all, whereas the control group contained one unovulated female (Fig. 6). There were no significant differences between the groups in the relative weight of stripped eggs. However, the value for the control group and also in pH 5.25 was lower than in pH 5.25+Al and pH 4.75 (Table 5), apparently because most females that were not already spent in these groups had at least partially released their eggs before stripping (Fig. 6). The low proportional weight of stripped eggs in pH 4.75+Al was, by contrast, due to a low ovulation rate. At stripping, the weight of ovaries (GSI) in pH 4.75+Al was significantly greater than in the controls (Fig. 2) due to the considerable number of unovulated eggs in the

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261

Fig. 3. Oocytes in the control vendace and those exposed to low pH with or without added Al (200 mg=7.4 mmol L
1) in September, 2 months before spawning, and after 66 days of exposure. The developmental stages of oocytes from A to D/E and atretic (Atr.) ones, classified according to Zawisza and Backiel (1970), are marked.

ovaries of these fish (Fig. 6). The fresh and dry weights of the stripped eggs were quite similar in all the groups, but the water content of eggs was lowest in pH 4.75+Al (Table 5). 4.4. Blood glucose and lactate The blood glucose concentration of females and males increased significantly (Po0:05) from September to October in all exposure groups, except for males in pH 5.25. In November, the blood glucose concentration in males was significantly (Po0:05) higher in all groups than in September. In females the blood glucose concentration in pH 4.75+Al from September onward was significantly higher than in the controls (Fig. 7); according to two-way ANOVA, acidity in September and acidity (Po0:001) and Al (Po0:05) in October significantly increased the blood glucose concentration of females (Fig. 7). Due to great internal variation, the blood glucose concentration of males was significantly higher than in the control only in November in pH 4.75 and pH 4.75+Al (Fig. 7), but according to two-way

ANOVA it had already increased significantly due to acidity in October; by November, acidity (Po0:001) and Al (Po0:05) had elevated the blood glucose of males (Fig. 7). There were no significant differences between the groups in blood lactate concentrations of either females or males (one-way ANOVA), although two-way ANOVA showed that in November the blood lactate concentration of males had increased (Po0:05) due to acidity (data not shown). 4.5. Plasma ions There were no significant differences in the plasma Ca2+ concentration of females between the groups, but both in September and October the mean value was highest in pH 4.75+Al (Fig. 8). There were, however, no significant effects of exposure in two-way ANOVA. Plasma [Ca2+] of males in some cases differed significantly between the exposure groups with the values tending to be highest in pH 4.75 in October (Fig. 8). The female-to-male ratios of plasma [Ca2+], calculated from

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Fig. 4. The testes of control vendace and those of fish exposed to low pH with or without added Al (200 mg=7.4 mmol L
1) in September, after 66 days of exposure, in October (108 day), approximately 2 weeks before the normal spawning time, and in November (145 day), 3 weeks after spawning. An arrow marks spermatozoa.

the group means, were highest in the pH 4.75 groups in September and in the pH 5.25 groups in October (Table 6), the latter effect resulting from the low plasma [Ca2+] of males in both pH 5.25 groups (Fig. 8). The differences in plasma [Mg2+] between the groups (data not shown) followed similar patterns in both sexes in September and October. After spawning in November, the plasma [Mg2+] of males was the lowest in the Alexposed groups (two-way ANOVA, Po0:001). In September, after 66 days of exposure, the plasma Cl
concentration of females, but not that of males, was significantly (Po0:05) lower in pH 4.75 and pH 4.75+Al than in the control. At this time no significant differences in the plasma [Na+] of females or males were detected between the treatments (Fig. 8). However, according to two-way ANOVA, acidity reduced (Po0:001) the plasma [Cl
] of females, and acidity and Al jointly (Po0:05) reduced that of males. About 2

weeks before spawning time, in October, there were no significant (P40:05) differences in the plasma [Na+] or [Cl
] of males between the exposures (Fig. 8), apparently due to the small sample size and large internal variation, but according to two-way ANOVA both these ion concentrations had decreased significantly (Po0:05) due to acidity. At that time, in females both plasma [Na+] and [Cl
] in pH 4.75 and pH 4.75+Al, and also [Cl
] in pH 5.25+Al were significantly lower compared to the control (Fig. 8); the decrease in the plasma [Na+] and [Cl
] was mainly due to acidity (Po0:001; two-way ANOVA) and in the [Cl
] also due to Al (Po0:05). By the November sampling, about 3 weeks after the normal spawning time, the plasma [Na+] and [Cl
] of males had considerably decreased in pH 4.75+Al and markedly in pH 4.75 (Fig. 8). Moreover, in pH 5.25+Al the plasma [Cl
] was significantly lower than in the control. Plasma [Na+] and [Cl
] in males in November were highly

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significantly (Po0:001) reduced by acidity and Al and, moreover, pH and Al jointly caused a moderate (Po0:01) reduction in the Cl
concentration.

263

Control (N = 23) 48%

1 2 3 4 5 6 7

4.6. Hematology In females there were no significant differences between the groups in September and October in the hemoglobin concentration (Hb), the hematocrit value (Hct), or the mean corpuscular hemoglobin concentration (data not shown), but according to two-way ANOVA, acidity decreased both Hb and Hct of females in September and MCHC in October. In males, MCHC in pH 4.75+Al was significantly (Po0:05) lower than in the control in September, and in November Hct was significantly higher in pH 4.75+Al than in the control (data not shown). Two-way ANOVA showed that Al decreased Hb

4% 4% 11%

33%

pH 5.25

pH 5.25+Al

(N = 20)

( N = 11) 8%

44%

17%

33%

17%

4% 4% 4%

8%

26%

33%

pH 4.75

pH 4.75+Al

(N = 11) 27%

( N = 11) 14% 21% 14%

9% 27% Fig. 5. The presence of spermatozoa in the testes (cf. Fig. 4) of control vendace and in those of fish exposed to low pH with or without added Al (200 mg=7.4 mmol L
1) in September, after 66 days of exposure, in October (108 days), approximately 2 weeks before the normal spawning time, and in November (145 day), 3 weeks after spawning. The extent of the regression of testes is also described. Arbitrary units were used: in September and October the scale was from 1 (=no spermatozoa) to 6 (=numerous spermatozoa) and in November from
8 (=no regression, numerous spermatozoa) to 12 (=advanced regression, no spermatozoa left).

18% 9%

9%

50%

Fig. 6. The proportions of female vendace (%) classified according to their ovulation rate during the normal spawning time (3–5 November) after exposure for 121 days (from late endogenous vitellogenesis) to pH 4.75 and 5.25 with or without added Al (200 mg=7.4 mmol L
1). The scale was 1 (=completely unovulated), 2–5 (=increasing degree of ovulation), 6 (=completely ovulated), and 7 (=spent, i.e., eggs already released).

Table 5 The proportional total weight of stripped eggs, fresh and dry egg weight, and egg water content of vendace

Total weight of eggs (%) Egg fresh weight (mg) Egg dry weight (mg) Water content of eggs (%)

Control

pH 5.25

pH 5.25+Al

pH 4.75

pH 4.75+Al

10.171.9 (23) 1.6970.08 0.5270.02 70.270.5 (12)

11.072.1 (20) 1.6770.08 0.4870.02 72.270.4 (14)

12.971.6 (11) 1.6170.12 0.4770.04 71.670.5 (9)

14.272.5 (11) 1.7170.09 0.5270.04 71.270.6 (10)

9.172.9 (11) 1.6870.07 0.5070.01 69.871.0 (10)

Note: Values are means7SE. At stripping fish had been exposed for 121 days to two pH levels with or without added Al (200 mg=7.4 mmol L
1). The numbers of sampled fish are in parentheses.

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Fig. 8. The mean (7SE) plasma Ca2+, Na+, and Cl
concentrations of female and male vendace in the control and treatment groups in September, October, and November, i.e., after 66, 108, and 145 days of exposure. For further details see the legend for Fig. 7. Fig. 7. The mean (7SE) blood glucose concentrations of female and male vendace exposed to pH 4.75 and 5.25 with or without added Al (200 mg=7.4 mmol L
1) from late endogenous vitellogenesis through the normal spawning time and sampled in September, October, and November, i.e., after 66, 108, and 145 days of exposure. The number of samples is given in parentheses and significant differences (Po0:05; Tukey’s test) between the treatments at each sampling time are indicated by dissimilar letters above the columns.

(Po0:05) and acidity MCHC (Po0:01) in males in September. In October the exposures had no significant effects on the hematology of males, but in November acidity significantly increased both the blood Hb and Hct and, moreover, acidity and Al jointly affected Hct. 4.7. Gill histology The gills of the control vendace had a typical appearance for salmonid fish. The lamellae of control fish became thicker (Po0:05) and those of acid-exposed fish tended to become thinner by the November sampling, but otherwise there were no major changes in the thickness of the lamellar epithelium in the control fish between the sampling times. The lamellae in pH 5.25

Table 6 The ratio of female-to-male plasma Ca2+ concentrations in vendace Control pH 5.25 pH 5.25+Al pH 4.75 pH 4.75+Al September 1.40 October 1.23

1.47 1.73

1.31 1.73

1.67 1.24

1.57 1.45

Note. Fish were exposed in two pH levels with or without added Al (200 mg=7.4 mmol L
1) and sampled in September and October, i.e., after 66 and 108 days of exposure, respectively. Numbers of females and males are given in Fig. 8.

in November were significantly thinner than in the control or pH 5.25+Al, and the lamellae in pH 4.75 with or without Al were also thinner, although not significantly, than those of control fish (Fig. 9a). Twoway ANOVA indicated that aluminum significantly increased the thickness of the lamellar epithelium in November (Po0:01). A few fish in pH 4.75+Al had extensively hypertrophied bases of the lamellae in October and November (Fig. 10). No separation of the outer epithelial layer occurred in any exposure group. As with changes in lamellar epithelium thickness, the thickness of the filament epithelium in the control group increased significantly by November, whereas in pH 5.25

P.J. Vuorinen et al. / Ecotoxicology and Environmental Safety 54 (2003) 255–276

265

(a)

(b)

Fig. 9. The mean (7SE) thickness of the gill lamellar (a) and filament (b) epithelium of vendace in the control and treatment groups in September, October, and November, i.e., after 66, 108, and 145 days of exposure. Number of observations was five (N ¼ 5) in every case. For further details see the legend for Fig. 7.

without Al it became significantly thinner from September to October (Fig. 9b). There was a slight trend for the thickness to increase from September to November in pH 4.75 and pH 4.75+Al, but this was not significant. In September, the filament epithelium was significantly thinner in pH 4.75 and in October in pH 5.25 than in pH 5.25+Al (Fig. 9b). According to a two-way ANOVA, both pH and Al significantly (Po0:05) affected the thickness of the filament epithelium in September but only Al in October (Po0:01) and November (Po0:001). However, it was clear at every sampling time that the filament epithelium was thinner in acidic water without added Al than in acidic water with Al. Aluminum was detected inside the gill epithelium only when the metal was added to the water. Most Al was found in the filament epithelium and occasionally in the lamellae (Fig. 11). The number of Al deposits was significantly higher in November compared to September in pH 4.75+Al (Fig. 12).

Fig. 10. The bases of the lamellae of vendace gills (hematoxylin-eosin staining) were hypertrophied following exposure to pH 4.75 and aluminum (200 mg L
1) in November (a). The gills of the control fish had a normal appearance (b).

The number of mucous cells per lamella, both in pH 4.75+Al and in pH 4.75, was higher than in the control group in September. This difference between the control and pH 4.75 was greatly reduced in October due to the decrease in mucous cell number in pH 4.75, but in pH 4.75+Al mucous cell hyperplasia was still significantly more frequent in October. In November the number of mucous cells in pH 4.75+Al had also reduced to the control level (Fig. 13). According to two-way ANOVA, pH affected the mucous cell numbers in September (Po0:001) and Al in November (Po0:05). Hypertrophy of mucous cells increased among control fish from September and October toward November, so that only one fish had enlarged mucous cells in September (N ¼ 10), none in October (N ¼ 8), but 61% (N ¼ 18) in November (Table 7). A less pronounced increase was seen in the other groups. In pH 4.75+Al, the gills already had mucous cell hypertrophy in September.

5. Discussion 5.1. Water chemistry The pH values and Al concentration to which the vendace were exposed corresponded rather well to those

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Fig. 11. Aluminum was mainly found inside the gill filament epithelium of vendace (arrow) following exposure to pH 4.75 (and 5.25) in the presence of added aluminum (200 mg L
1). Gills were stained with hematoxylin sodium iodate.

Fig. 12. The number of Al spots per lamella (mean7SE) in the Alexposed vendace at different sampling times. The statistical differences (Po0:05; Tukey’s test) between groups within the given month are denoted with letters, the same letter indicating no difference. The statistical differences between the months within the exposure groups are denoted with an asterisk. The number of fish is given in parentheses.

found in ‘‘whitefish lakes,’’ i.e., acidified lakes, supporting whitefish populations in a field survey (Vuorinen et al., 1994). The mean pH in these whitefish lakes was 5.69 with a minimum pH of 4.55 and the mean total Al concentration was 99 and maximum 201 mg L
1. Although the experimental lake water is considered soft, its Ca2+ concentration (0.23 mmol L
1) was 3.5 times higher than the mean in the acidified ‘‘whitefish lakes.’’ The decrease in the [Ca2+] has been demonstrated to increase the toxicity of acidic waters (Booth

Fig. 13. The number of mucous cells (mean7SE) in the gills of vendace exposed to 0 or 200 mg L
1 of added Al. For further details see the legends for Figs. 7 and 12.

et al., 1988; Wood, 1989), because Ca2+ stabilizes the respiratory membrane and thereby reduces its electrolyte permeability (Wood, 1989) and, on the other hand, H+ and probably also Al3+ compete with Ca2+ for its binding sites (see Gensemer and Playle, 1999). For example, survival and growth of brook trout were better in exposure to pH 5 with or without 170 mg Al L
1 in water with a [Ca2+] of 0.2 than with 0.01 mmol L
1, but no differences in osmoregulation were observed between these Ca2+ concentrations (Mount et al., 1988a, b).

P.J. Vuorinen et al. / Ecotoxicology and Environmental Safety 54 (2003) 255–276 Table 7 The percentage of vendace exhibiting mucous cell hypertrophy September

Control pH 5.25 pH 5.25+Al pH 4.75 pH 4.75+Al

October

November

%

N

%

N

%

N

10 0 40 20 91

10 8 10 10 11

0 33 50 43 43

8 6 8 7 7

61 38 81 47 62

18 16 16 15 13

Note. The midsection of gill filaments was excised following exposure at pH 4.75 and 5.25–0 and 200 mg L
1 added Al. N is the number of fish.

Whitefish yolk-sac fry were most tolerant of acidity in different Al concentrations in test solutions ([Ca2+] 0.04–0.12 mmol L
1) with the highest [Ca2+] but also containing some dissolved organic carbon (DOC) (Vuorinen et al., 1994; Kein.anen et al., 1998). However, the [Ca2+] of the test water in the present study exceeded those effective low [Ca2+] and probably did not affect toxicity. The test solutions were fresh with respect to Al since the acidic Al stock solution was continuously mixed with the incoming lake water with a 3-h 90% replacement time. The reactions of Al with ligands and against pH and temperature changes are very rapid (Lydersen, 1990). Organic and fluoride complexes are in general the predominant Al forms in acidified surface waters, and at pH below 5.5 all the water F
is complexed with Al (Gensemer and Playle, 1999). Organically complexed Al is only slightly toxic to fish (Spry and Wiener, 1991). On the basis of the color value, the DOC of the L. Yl.aEnonvesi water was low, about 4 mg L
1. According to theoretical calculations, even this low DOC complexed a considerable part of the Al, especially at pH 5.25 (see also Spry and Wiener, 1991). Nevertheless, fluoride complexes were the dominant species in the fast-reactive Al at the lower test pH, whereas only 5–15% of Al was present as free ion. According to Wilkinson et al. (1990), the free Al3+ ion and the fluorocomplex AlF2+ were the most toxic forms to Atlantic salmon (Salmo salar) parr. Gensemer and Playle (1999) proposed that the toxicity of the fluorocomplex would result from the gills outcompeting this complex for Al. 5.2. Mortality At each exposure pH the mortality rate of vendace was higher with added Al than without it; the acidity itself at pH 5.25 did not increase mortality. For the whitefish, which is approximately as acid-tolerant as vendace, the threshold values of effective pH, based on yolk-sac fry mortality and swimming activity, were determined to be 4.5–5.5 depending on the Al

267

concentration in water with a [Ca2+] of 0.12 mmol L
1 (Vuorinen et al., 1993). The mortality of adult whitefish was also somewhat higher at pH 4.75 when 150 mg L
1 of Al was added (Vuorinen et al., 1990). Accordingly, the nominal Al concentration was the best explanatory variable of survival in the long-term exposure of brook trout to low pH and Al in various Ca2+ concentrations (Mount et al., 1988b), and the brook trout mortality rate increased with an increase in Al concentration (Booth et al., 1988). Mortalities occurred in brook trout exposed for 10 weeks to pH 5.2 only if they were simultaneously exposed to Al with low (0.05 mmol L
1) water [Ca2+] (Wood et al., 1988). In the present study, the pH (4.75) in which the highest mortality occurred in the presence of Al (200 mg Al L
1) was in any case higher than the acutely lethal values for newly hatched vendace for which the 96-h LL50 pH value was 4.4 at an Al concentration of 200 mg L
1, and the incipient LC50 value of Al was 400 mg L
1 at pH 4.5 for one-summerold whitefish (Rask et al., 1988). The lethal level would be expected to be lower in a long-term exposure, although older fish are generally more tolerant of toxicants than those in early life stages. The mortality of adult whitefish due to exposure at pH 4.75 with 150 mg Al L
1 was, however, considerably lower (17.5%) (Vuorinen et al., 1990) than in vendace at pH 4.75 with 200 mg Al L
1. The rather high overall mortality rate during the first two exposure months is perhaps explained by unnatural circumstances in the test basins, since vendace prefer deeper and cooler waters during daytime at the elevated summertime water temperatures. The stressful conditions were seen as elevated mortality rates in the control at water temperatures over 15 C, whereas in cooler water the mortality rate smoothed. However, such smoothing was not detected in the pH 4.75 groups and pH 5.25+Al. In these exposure groups, high mortality seemed to be connected with physiological stress caused by low pH and Al that appeared as a poor appetite and lower condition factor of the fish. The dead fish were even leaner toward the end of the exposure in these groups. At the lower exposure pH the mortality rate of males was higher than that of females, and increased at pH 4.75+Al most sharply after spawning when the water temperature had decreased steeply from 5 C to 2 C. This was not as obvious in females, as nearly all females had been removed at the stripping of eggs about 1 week earlier. The reason for this mortality increase was probably not the change in Al chemistry, as according to the theoretical calculations there were only minor differences in the speciation of Al between these temperatures. However, the accumulation of Al inside the gill tissue was more pronounced by November, which was suggested to be related to changes in the gill membranes. The dramatic decrease in plasma [Na+] and [Cl
] and the increase in blood glucose concentration in

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vendace up to the November sampling indicate severe ionoregulatory disruption that apparently caused the high mortality. 5.3. Growth The acidity at pH 4.75 and Al at each pH level affected the appetite of vendace, which did not take feed as willingly as fish in the control and at pH 5.25, and evidently as a result of this they were lean. Similarly, whitefish were lean after 143 days of exposure to pH 4.75 with or without added Al (Vuorinen et al., 1990). The sublethal exposure of salmon, brown trout (Salmo trutta), brook trout, and rainbow trout (Oncorhynchus mykiss) to low pH with or without Al has been demonstrated to impair their appetite and lead to diminished growth (Tam et al., 1988; Mount et al., 1988a, b; Wilson et al., 1994). According to Mount et al. (1988b), the growth of brook trout was retarded in those combinations of pH, Al, and various [Ca2+] where survival also decreased, and the decreased growth was related only to Al and Ca2+, with Al explaining most of the variance. Vendace grew somewhat during the exposures as the mean length and weight of all sampled females and males were larger than the respective initial values. The mean increase in female weight was 14% and in males 2%; in females the greater part of the increase was apparently due to the growth of gonads. These are very low figures compared with whitefish exposed in comparable conditions where the weight of both females and males increased by about 90% (Vuorinen et al., 1990). Whitefish received a ration of 4% day
1 whereas the amount given to vendace was 2% of wet body weight per day. Feed was offered twice a day, whereas in natural conditions, vendace feed practically continuously. Wilson et al. (1996) observed that rainbow trout exposed to Al at pH 5.2 decreased feed intake during the first week but then recovered. In that experiment the Al concentration was very low, only 30 mg L
1, so the observed effect could have been mainly due to the initial effect of low pH. The feeding of lake whitefish (C. clupeaformis) decreased in an exposure to pH 4.7 and even ceased at a lower pH (Scherer et al., 1986). On the contrary, Tam and Payson (1986) observed no differences in brook trout feed intake in an exposure to pHs 4.5, 5.0, and 5.5, but nevertheless the growth of exposed fish was reduced compared to the control. The authors concluded the growth inhibition to be due to the low pH, which had disturbed the metabolism of the brook trout. Wilson et al. (1994), by contrast, concluded that decreased growth of rainbow trout exposed to Al at pH 5.2 was specifically caused by loss of appetite and not by metabolic changes. Decreased appetite in low pH exposures has been suspected to be due to increased blood or plasma glucose concentration, the so-called

glucostatic theory (Scherer et al., 1986). In the present study the appetite of vendace at pH 4.75 was low all the time, but the blood glucose was only somewhat elevated after 66 days of exposure and markedly increased after the 108th day onwards. Sustained increased cortisol excretion has been reported to decrease appetite, growth rate and food conversion efficiency (Gregory and Wood, 1999). Nevertheless, the increase in mortalities, increase in the blood glucose concentration, and decrease in the plasma [Na+] and [Cl
] of vendace coincided towards the end of the exposure and, along with the severity of the exposure, peaked at pH 4.75+Al. 5.4. Reproduction The GSI of female fish increases particularly during exogenous vitellogenesis, i.e., toward spawning. This phase of the reproductive cycle in vendace occurred between the first and second sampling, from September to October, and appeared in the severalfold increase in the GSI of females that was most marked in the control group. Low pH and Al clearly disturbed the development rate of oocytes, which was apparent as a lower GSI than in the control in October after 108 days of exposure, 2 weeks before the spawning time. By September, lower proportions of more advanced oocyte developmental stages were already seen in all exposure groups. The proportional numbers of atretic oocytes in vendace were quite similar in the treatment groups and the control in September, 2 months before spawning. Tam and Zhang (1996) were also unable to detect differences in the number of atretic oocytes between acid-exposed and control brook trout during the later period of oocyte development. However, it is possible that atresia might have mainly occurred during the earlier period of oocyte development in vendace. Tam et al. (1990) observed increased numbers of atretic oocytes during early vitellogenesis in brook trout that were exposed to pH 4.5, but later there were no differences between the acid-exposed and control fish. Disturbed oocyte development, or at least a delay in the final maturation, was revealed by the large proportion of unovulated yolky eggs in the ovaries in the most severe exposures, peaking in pH 4.75+Al, when the ovaries of the vendace were examined at the spawning time after stripping of eggs. Unovulated oocytes were also observed in brook trout exposed to low pH and low Ca water (Mount et al., 1988a) and in whitefish exposed to pH 4.75 without, but especially with, added Al at 150 mg L
1 (Vuorinen et al., 1990), or to pH 5 with 100 mg Al L
1 (Vuorinen and Vuorinen, 1991). After a 10-month exposure to pH 4.5, the ovulation of brook trout was delayed by several days compared to the control group (Tam and Payson, 1986), and brook trout exposed to low pH+Al in low Ca water tended to mature later than control fish, but all fish matured

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within 2 weeks (Mount et al., 1988a). However, the authors reported that these fish still had unovulated eggs after spawning. The ovulation of whitefish also occurred later at pH 4.75 with or without added Al, and even at pH 5.75 with 150 mg Al L
1, than in the controls (Vuorinen et al., 1990). In that experiment, 28% of whitefish females exposed to pH 4.75 with Al remained completely unovulated when examined approximately 3 weeks after their spawning period, 4 weeks from the beginning of spawning in the control group, whereas all the control fish had ovulated within a week. Perhaps they would not have spawned at all, which might also have been the case with a number of vendace in pH 4.75+Al. Beamish et al. (1975) reported that in an acidified lake, 65–75% of white sucker (Catostomus commersoni) females failed to release their ova and several other fish species had not spawned when examined after their spawning season. It is therefore possible that ovulation was not only delayed but completely arrested, although the authors did not report the timing of their investigations exactly. Ovulation in whitefish was slightly delayed even at pH 6.0 when 100 mg Al L
1 was added to the same dilution water as in the present study, but under such mild exposure conditions ovulation was not prevented (Vuorinen and Vuorinen, 1991). Acid exposure reduced daily vitellogenin production in rainbow trout (Roy et al., 1990). No significant difference was recorded between the treatments and control in the fresh or dry weight of those vendace eggs that were mature at the normal spawning time, indicating the normal yolk deposition and growth of these oocytes. Opaque and clotted eggs, which had not undergone final maturation and were therefore difficult to strip, were not included in the measurements. Acid exposure did not affect the quality or viability of the mature eggs of either brook trout or whitefish (Mount et al., 1988a; Vuorinen et al., 1990; Vuorinen and Vuorinen, 1991; Tam and Zhang, 1996). Egg size of acid-exposed brook trout was similar to that in control fish (Tam and Zhang, 1996), but exposure of whitefish to acidity with or without Al resulted in a lower egg dry weight (Vuorinen et al., 1990). In general, oocytes absorb quite a lot of water immediately before ovulation, but the eggs of vendace in pH 4.75+Al contained less water than the eggs of control females. In whitefish, no significant differences were detected between treatments in egg water content (Vuorinen et al., 1990), but whitefish were, unlike vendace in this study, stripped of mature eggs over a longer period of time as they ovulated. In rainbow trout exposed to acidic water for 1–2 weeks before ovulation, plasma concentrations of the ovulation-inducing steroid hormone, 17a,20b-dihydroxy-4-pregnen-3-one, were lower than normal, on the basis of which low pH was concluded to affect the final maturation of oocytes through disturbance of

269

endocrinological mechanisms (Ikuta and Kitamura, 1995). The effect of disturbed water balance, which was observed as an increase in blood viscosity, in vendace at pH 4.75 remains open to question as a cause of the inhibition of water uptake by oocytes during their final maturation. There were no clear effects of acidity and Al on the plasma [Ca2+] in females or males before spawning. The female-to-male plasma [Ca2+] ratio was 1.23 in the control about 3 weeks before the spawning time, and in pH 4.75+Al it was 1.45, although a considerable proportion of these females remained unovulated. The suggestion of Beamish et al. (1975) that this ratio should be larger than 1.4 for successful spawning was not evident in vendace. Furthermore, the ratio was 1.48 in whitefish exposed to pH 4.75, and of these fish 8% remained unovulated (Vuorinen et al., 1990); in another experiment in whitefish whose ovulation was delayed, the ratio was 1.75 following exposure to pH 5 and Al (Vuorinen and Vuorinen, 1991). However, in perch, another species whose spawning was found to be delayed in acidified lakes, the female-to-male ratios for plasma [Ca2+] were 1.00–1.32 as compared with 1.64 in a circumneutral lake just after the normal spawning time (Vuorinen et al., 1992). Vendace clearly showed clinical signs of stress, seen as markedly elevated blood glucose concentrations, especially in pH 4.75+Al. At the same time, the plasma cortisol concentration had obviously increased. Cortisol production is the primary response to stress in fish (Mazeaud et al., 1977) and plays an important role in providing fuels such as glucose to maintain homeostasis (van der Boon et al., 1991). According to Leatherland (1999), hypercortisolism is a normal characteristic during late gonadal maturation in many salmonids. Reddy et al. (1999) found that cortisol had no effect on the steroid 17,20b-dihydroxy-4-pregnene-3-one in postvitellogenic rainbow trout oocytes in vitro. However, in rainbow trout that were repeatedly subjected to acute stress during 9 months prior to spawning, the plasma cortisol concentration increased after stress, and ovulation was delayed, but no effects were observed on fecundity; nevertheless, eggs were smaller and the body weight of stressed fish tended to be lower (Campbell et al., 1992). It is possible that a stress-induced increased cortisol exerts its effect earlier during oocyte development, during mid- and peak vitellogenesis. This is supported by the fact that in the present study, in September to October, the GSIs of female vendace in the exposure groups were lower than in the control. Cortisol was also demonstrated to significantly suppress gonadotropic hormone- (GtH)-stimulated testosterone and 17bestradiol production in peak vitellogenic follicles from rainbow trout in vitro (Reddy et al., 1999). Nevertheless, in his review of the effects of stress on the reproduction of fish, Wendelaar Bonga (1997) came to the conclusion

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that the corticosteroid response in salmonid species is attenuated during the reproductive period. In contrast, in their review of cortisol in teleost fishes, Mommsen et al. (1999) stated that glucocorticoids suppress reproductive functions and form a part of the mechanism delaying reproduction under stressful conditions. Apparently, more information is required about the effects of long-term, sustained stress during the whole cycle of vitellogenesis. Consistently with the earlier maturation of oocytes, spawning behavior in vendace started 1–2 weeks earlier in the control than in the pH 4.75 treatments, which also indicates retarded oocyte development or delayed maturation due to low pH and Al. Kobayashi et al. (1986) showed that the presence of ovulating female goldfish (Carassius auratus) was necessary for the gonadotropin surge and spawning behavior in males. In acidified lakes the spawning of perch was detected to occur later than in circumneutral lakes (Rask et al., 1990; Vuorinen et al., 1992); in one severely acidified lake (pH about 4.5, Al about 12 mmol L
1), perch spawned as much as 1 month late, and all of the eggs found at spawning sites were dead. In vendace, the low proportional weight of stripped eggs reflects in pH 4.75+Al a delayed oocyte final maturation and ovulation rate. However, the number of stripped eggs was lowest in the control group followed by the pH 5.25 and pH 5.25+Al groups, due to the release of eggs in these groups before the inspection of ovulation and stripping of eggs. Eggs were released in spontaneous spawning activity depending on the ovulation rate. Therefore, the mean proportional weight of eggs, even in the control group, in the present study was considerably lower than values of 26–27% reported for vendace from a lake in central Finland (Lahti, 1991). However, the values of five control fish were 24–26% and, overall, the GSI of 22% for the control vendace in October could have resulted in a value close to that reported by Lahti (1991). In the exposure of whitefish (Vuorinen et al., 1990) there were no significant differences in the proportion of eggs at stripping between the different exposure pH (4.75 and 5.75) with or without added Al (150 mg L
1). This was likewise apparently due to spontaneous partial spawning of whitefish in the control and the mildest exposure groups. In pH 4.75+Al as many as 50% of vendace females were totally unovulated at the normal spawning time but the delay in ovulation was also obvious in pH 4.75 without Al and pH 5.25 with Al if the proportions of females that had retained the majority of their oocytes in an unovulated state is taken into account. In male vendace, the delay in spermatogenesis was seen as a tendency toward a lower GSI in exposure groups in September and October. However, although both acidity levels retarded spermiogenesis in September, 2 months before spawning time, the delay was not

so obvious if Al was present. Reduced androgen production might be behind the retarded spermiogenesis detected in vendace in October. In ascending salmon the plasma testosterone and 11-ketotestosterone levels were significantly lower in an acidic (pH 4.7) than in a less acidic (pH 5.6) river water (Freeman et al., 1983). After the spawning season the retardation in male reproductive physiology was apparent as a longer milt running time in pH 4.75+Al than in the control and as a GSI that was still high about 3 weeks after the normal spawning time. The delayed regression of testes was also found in whitefish after exposure for 4 months to low pH with or without Al (Vuorinen et al., 1990; Vuorinen and Vuorinen, 1991), and in perch from acidified lakes (Vuorinen et al., 1992). Pheromonal signals from females (Olse! n and Liley, 1993) with delayed ovulation probably induced sustained milk production in vendace males and postponed the regression of testes. 5.5. Blood glucose and lactate As indicated by the blood glucose concentrations, which had slightly increased according to the severity of exposure conditions already in September and further increased throughout the exposure months, vendace were clearly experiencing stress from the exposures to low pH and Al. Elevation of the blood glucose concentration, induced by corticoids and catecholamines, is a well-known secondary response to stress in fish (Mazeaud et al., 1977; Wendelaar Bonga, 1997). Plasma cortisol and glucose concentrations in rainbow trout increased during a 21-day exposure to pH o5.2 (Brown et al., 1984). It is not known how the plasma cortisol concentration behaves if an exposure to low pH and Al persists for months. Elevated blood or plasma glucose concentrations in long-term acid exposures have been detected in brook trout, rainbow trout, and whitefish (Tam et al., 1987; Vuorinen et al., 1990; Vuorinen and Vuorinen, 1991). As in female vendace, the plasma glucose concentration in female brook trout exposed to pH 4.5 for 10 months tended to rise more than in the males (Tam et al., 1987). Similar observations were also made in whitefish exposed for over 4 months to pH 4.75 and 5.0 with or without added Al (Vuorinen et al., 1990; Vuorinen and Vuorinen, 1991). In the present study the highest blood glucose concentrations were measured in males in pH 4.75+Al at the time period of high mortalities in November when females were not sampled. The blood glucose concentration seemed also to increase in the control vendace as the exposure progressed. Elevated plasma cortisol is a normal characteristic exhibited by many salmonid species during late gonadal maturation (see Leatherland, 1999). Although the elevation of blood glucose concentration is a stress response (Mazeaud et al., 1977;

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Wendelaar Bonga, 1997), it is apparently a means to maintain plasma osmolality after an electrolyte decrease . (Bergstrom, 1971; Brown et al., 1984; Scherer et al., 1986; Audet et al., 1988). Giles et al. (1984), when exposing rainbow trout for 22 days to different pH levels and measuring plasma osmolality, concluded that plasma contained some unidentified factor that increased osmolality by 40% in fish exposed to low pH. . These authors calculated from the data of Bergstrom (1971) that a 3.4-mol decrease in plasma [Na+] and [Cl
] corresponds to an increase of 1 mol in blood glucose concentration. Using these values the increased blood glucose concentration of female vendace in the treatment groups accounted for 21
29% of the decrease in plasma Na+ and Cl
concentrations in October; in males there was more variation so that in October the compensations were 24
34% and in November 10
29%. Nonetheless, in vendace the highest blood glucose levels were detected coincident with large decreases in the plasma [Na+] and [Cl
]. In rainbow trout exposed for 2 weeks to pH 4.2 (which was apparently lethal to this acid-sensitive species, although authors did not report mortalities), plasma osmotic pressure decreased despite an increase in the plasma glucose concentration (Lee et al., 1983). In that experiment the rise in the plasma glucose level was roughly only twofold and in whitefish two- to threefold (Vuorinen et al., 1990), whereas the increase in the blood glucose concentration of vendace was five- to sixfold at its greatest. A similar large increase was seen in rainbow trout exposed for 3 weeks to pH 4.7 (Brown et al., 1984). The blood lactate concentrations in female and male vendace were in general lower than in whitefish (Vuorinen and Vuorinen, 1991). The fact that there were no significant differences between the treatments and the control and that the concentrations were low indicates mainly that the sampling procedure did not cause extra stress to fish (Oikari and Soivio, 1975) or at least affected them all in a similar way. 5.6. Plasma sodium and chloride During the warm water temperatures in summer, no samples were taken in order to avoid additional stress to the fish. Nevertheless, in September, after 66 days of exposure, only the plasma Cl
concentration of females in both pH 4.75 groups had significantly decreased. It is possible that the ionic losses of vendace were then compensated through diet (2% day
1), similarly to juvenile rainbow trout exposed to pH 5.2 and fed a ration of 1% day
1 or greater that showed no ionoregulatory disturbance (Morgan et al., 2000). According to Smith et al. (1989), sodium intake from food is as great as branchial intake during summer time, and at that time of the year the food intake of vendace is also highest (Lahti and Muje, 1991).

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It is also possible that vendace adapted physiologically to some degree during the first 2 months of exposure. Plasma Cl
and Na+ levels of juvenile rainbow trout recovered after 35 days of exposure to pH 5.2 with or without a low concentration (38 mg L
1) of Al (Wilson et al., 1994). In addition, brook trout were shown to acclimate to acid/Al stress (pH 5.2, 75, and 150 mg Al L
1) during a 10-week exposure (Wood et al., 1988). Adaptation may result from adjustments in ionic regulation, as in a 3-month sublethal exposure to acidity (pH 4.8) the Na+ and Cl
net fluxes of rainbow trout recovered, although the influx was still partially inhibited (Audet et al., 1988). By comparison, the Na+ influx of yolk-sac fry of acid-sensitive roach (Rutilus rutilus) was unable to recover after an exposure to pH 5.00 with an Al concentration of 200 mg L
1, but that of acid-resistant pike (Esox lucius) recovered even after an exposure to pH 4.00 (Kein.anen et al., 2000). Vendace is between these two species in sensitivity (Vuorinen et al., 1993, 1994). Despite feeding vendace with a daily ration of 2%, the decrease in plasma [Cl
] and [Na+] at pH 4.75 with or without Al continued from September onward. In October, after 108 days of exposure, the drop in plasma [Cl
] and [Na+] was approximately equal to that observed in brook trout in a 6-week exposure to Al at pH 5.2 (McDonald et al., 1991). In both pH 4.75 groups, the loss of plasma Cl
and Na+ in vendace males in October was approximately 11% compared with the control and in females about 10% in pH 4.75 and 17% in pH 4.75+Al. In a similar experiment to the present study, the plasma [Na+] and [Cl
] of both female and male whitefish was lower than in the control after a 143day exposure to pH 4.75, and in females the ion concentrations were lowest, consistent with the present study, in the exposure to Al (150 mg L
1) at this pH (Vuorinen et al., 1990). It should, however, be noted that the number of vendace males whose plasma [Cl
] and especially [Na+] was analyzed was low in some groups in October due to the small amount of sampled blood. The inability to maintain plasma ion balance from September onward may be related to the homeoviscose adaptation in gills to the cooling water temperatures. Another reason could be the proximity of the spawning time, which could have rendered vendace more sensitive to extra stress. In males in November, about 3 weeks after the spawning time at the end of the exposure (on days 141–145), the significantly lower plasma [Na+] and [Cl
] in both pH 4.75 groups and in pH 5.25+Al than in the control indicated severe disruption in ion regulation appearing as increased mortalities in these groups. In pH 4.75+Al especially, fatal loss of these plasma ions seemed to have continued after sampling in October, resulting in a 44% drop in the plasma [Cl
].

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5.7. Hematology The Al/acid exposure affected vendace hematology only slightly. Adult brook trout, which were exposed for 193 days to low pH at two Ca2+ levels with or without Al also showed no clear trend in Hct values during the experiment, apart from a decrease during the latter part of the exposure (Mount et al., 1988a). However, in that experiment the blood samples were stored on ice for half an hour before the Hct determination and such treatment changes fish blood properties considerably (Oikari and Soivio, 1975). In the present study, blood for Hct determinations was centrifuged immediately after sampling. In males the significant decrease in MCHC in the pH 4.75+Al group in September indicated the swelling of erythrocytes, because the Hct did not seem to decrease as much as the Hb concentration. This trend in MCHC was detected in nearly all exposure groups for both females and males after 66and 108-day exposures in September and October, respectively. The elevated Hct value in male vendace in November, when there were no significant differences in MCHC in pH 4.75+Al, might have been due to hemoconcentration, the withdrawal of water from plasma after an extensive plasma ion loss in these fish (Wood, 1989). Similarly, in brown trout exposed to acidified river water, the blood Hct value increased as plasma ion concentrations decreased (Muniz et al., 1987). Blood viscosity was not measured in vendace, but in November it was difficult to draw blood samples from vendace in pH 4.75 groups, as blood did not seem to be as freely flowing as during earlier samplings. Likewise, Muniz et al. (1987) found difficulties in blood sampling from brown trout exposed to acidified river water. In rainbow trout exposed for 22 days to acidities of pH 4.21
5.97 the plasma water content at pH 4.21 was lower than in the control (Giles et al., 1984). Hemoconcentration was not, however, detected in rainbow trout, although plasma ion levels dropped extensively and high mortalities occurred; instead, the blood Hb concentration and Hct value decreased as a function of a decreasing exposure pH, and MCHC decreased as well. 5.8. Gill morphology The gills of control vendace in the present experiment started to show thickening of lamellar and filament epithelium following the decrease in water temperature late in the autumn. A thickening of the epithelium during the cold water period in neutral water has also been observed in largemouth bass (Micropterus salmoides) (Leino and McCormick, 1993), which undergo an overwintering torpor, and in the rainbow trout, eel (Anguilla anguilla), and roach (H. Tuurala, pers. commun.) and may reflect the needs of fish to protect themselves against passive ion efflux while the active ion

uptake is slow due to the cold (Staurnes, 1993). This thickening does not occur in all fish species, however (see Leino and McCormick, 1993). Aluminum caused hypertrophy in the basal region of the gill lamellae in some but not all vendace at pH 4.75+Al in October and November, but not prior to this. The thickening in the basal region of lamellae in vendace suggests that it was mainly caused by the enlargement of chloride cells as a response to ion loss (cf. Laurent and Perry, 1991). However, this thickening of the basal region did not influence the measured thickness of the lamellae. Lamellar thickening has been observed in other studies following acute acid or Al exposure (e.g., Evans et al., 1988), but the time of exposure in the present experiment was probably long enough to allow the restoration of any major morphological changes that may have occurred in the gills. Fish gills are capable of recovering from the initial damage (Norrgren et al., 1991) with a concomitant increase in metal tolerance (McDonald and Wood, 1993). It is possible that the normal increase in the thickness of the lamellar epithelium was inhibited in the present experiment in acidic water but that exposure to Al reversed the thinning of filament epithelium caused by low pH alone back to the control level. Thickening of the filament epithelium is commonly observed in acute as well as long-term exposure to aluminum under laboratory conditions and in the wild (Vuorinen et al., 1990; Norrgren et al., 1991; Peuranen et al., 1993; Wilson et al., 1994). However, there are some reports of fish with only minor changes in the gill epithelium in waters with increased Al concentrations (Lacroix et al., 1990), one example being perch from acidified lakes (Vuorinen et al., 1992). 5.9. Aluminum accumulation in the gill tissue The incoming water had a background total Al concentration of about 6 mg L
1, which was largely bound to the organic matter in the water, at least at the higher test pH (see Section 5.1 and Spry and Wiener, 1991), and did not accumulate in the gill tissue in the non-Al treatment groups. The amount of Al in the gill tissue was not quantified in the usual way, i.e., as micrograms per grams, since the histochemical approach employed here avoids the confounding factor of Al binding to mucus on the gill surface. The histochemical detection of Al inside the gill tissue suggests that Al must have been in intimate contact with the epithelium and finally crossed the cell membrane (cf. Exley et al., 1991). In the gills of vendace this happened only in late autumn. The accumulation of Al mostly in the filament epithelium suggests that the metal was mainly taken up by chloride cells (KarlssonNorrgren et al., 1986; Norrgren et al., 1991). Aluminum in gills has been detected within chloride cells using

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microanalytical techniques (Karlsson-Norrgren et al., 1986; Norrgren et al., 1991) and is known to damage chloride cells directly (Evans et al., 1988). The gills of vendace most likely had time to recover from any initial damage (cf. McDonald and Wood, 1993) before the first sampling in September. In late autumn, however, Al tolerance apparently decreased, because the combination of low pH (especially pH 4.75) and Al disturbed ion regulation most dramatically from October to November. This ionoregulatory disturbance coincided with the presence of a large number of Al precipitates inside the epithelium in Al-exposed fish. Similar results have also been reported for whitefish: the number of Al precipitates correlated with the disturbances in ion regulation at pH 4.75 but not at pH 5.75 (Peuranen et al., 1993). In perch, however, the number of Al precipitates inside gill tissue did not correlate with ion regulation (Peuranen et al., 1993). Pole! o et al. (1991) concluded that a low water temperature reduces the acute toxicity of Al due to a slower polymerization. This effect may also reflect the lower metabolic rate of fish in the cold and hence their lower requirement for oxygen. Long-term exposure to Al in cold temperatures has nevertheless been associated with greater disturbances in ion regulation, a higher overwintering mortality, and pronounced gill damage. Karlsson-Norrgren et al. (1986) found more Al-induced gill lesions in brown trout at 2.5 C than at 15 C. They suggested that a ‘‘gill disease’’ reported during late winter on Swedish fish farms since the 1950s could be due to the effects of Al at water temperatures below 4 C. Both ionoregulatory disturbance and gill damage were also exacerbated by Al exposure in overwintering largemouth bass (Leino and McCormick, 1993). The greater Al accumulation in the gills of vendace during the cold-water period may be related to the combined effect of low temperature on membrane fluidity (see Hazel, 1979; Bolis et al., 1984; Exley et al., 1991) and an increase in the concentration of toxic forms of Al at low pH, although the latter was not supported by theoretical calculations with MINEQL+. Detailed studies that include these interactions would therefore be valuable to improve understanding of the effects of low temperature on Al toxicity. Moreover, changes in the physiology of vendace in preparation for spawning may have had some effect. Nevertheless, the abrupt increase in mortality in pH 4.75+Al coincided with the decrease in water temperature to about 2 C. 5.10. Gill mucous cells The number of mucous cells in gills at pH 4.75, with and without Al, was highest in September but reduced toward the control level at the end of the exposure period. Fischer-Scherl and Hoffmann (1988) observed an increase in the mucous cell number in brown trout

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gill during the snowmelt period in an acidified river, which had a total water Al concentration of 800 mg L
1. Mucous cell hyperplasia has also been observed in whitefish gills in acidified lakes (Peuranen et al., 1993). Since the high number of mucous cells may be a protective response against metals (Wilkinson and Campbell, 1993), it is possible that the increase in the number of Al deposits in November inside the gill tissue in the pH 4.75+Al treatment group was due to reduced mucus secretion in these fish.

6. Conclusion Results from vendace were consistent with the earlier results from a related species, the whitefish, showing that exposure to Al in acidic water delayed, or even prevented, the final maturation of oocytes and ovulation. This effect was also seen as delayed spawning behavior and delayed regression of testes in males after spawning. Stress, induced by exposure to low pH and especially to a combination of low pH and Al, apparently affected oocyte development earlier during vitellogenesis, which was seen as low gonadosomatic indices, and spermatogenesis tended also to delay due to acidity. Despite the effects on oogenesis, those eggs that matured in exposed vendace were viable, which is also consistent with earlier observations from whitefish. Under acidified conditions, the reproductive potential of vendace might be reduced even if mass mortalities of this species are not detected. Furthermore, a delay in spawning in the wild might postpone spawning until after lake freezing or postpone hatching of vendace to unfavorable times as regards food availability and competition with other species and may thereby reduce the survival of juveniles over their first winter. Aluminum clearly increased the toxicity of low pH that appeared in a disturbed ion balance, elevated blood glucose concentration, and even in mortality of vendace. Because Al decreased the appetite of vendace, especially at pH 4.75, increase in mortalities apparently resulted from the poor feeding of these individuals in addition to the differences in sensitivity between the fish. These detected responses were quite similar to those in earlier experiments on whitefish. As observed in the earlier studies, increased blood glucose concentrations coincided with the extensive plasma ion losses, suggesting the role of glucose in conserving the plasma osmotic pressure. Although vendace seemed to adapt to some degree to low pH with or without added Al in summer, the adaptation capacity broke down in the autumn, probably due to either the decreasing water temperature and concomitant decrease in feed intake or/and spawning stress. It also coincided with an increase in Al accumulation inside the gill tissue, suggesting changes in gill membrane properties. The vendace is adapted to a

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cold climate but it experienced problems with dissolved Al at low water temperatures. Thus, due to changes in physiology, vendace, and probably also other autumnspawning fish species, exhibit higher sensitivity near the spawning time than earlier in the season to conditions of acidification.

Acknowledgments The authors thank everyone who contributed to this research with their valuable assistance and to the preparation of this article with their comments. Thanks are especially expressed to the staff of the Finnish Game and Fisheries Research Institute at both Saimaa Fisheries and Aquaculture and at the laboratory in Helsinki and Mr. Hannu Revitzer, MSc., for aluminum analyses. Dr. Roy Siddall, Ph.D., checked the English. The study was financed by the Ministry of Agriculture and Forestry.

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