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The toxicity of aluminium to two acido-tolerant green algae
War. Res. Vol. 22, No. 8, pp. 977-983, 1988 Printed in Great Britain. All rights reserved
0043-1354/88 $3.00+0.00 Copyright © 1988 Pergamon Press plc
THE TOXICITY OF ALUMINIUM TO TWO ACIDO-TOLERANT GREEN ALGAE A. CLAESSON and L. T6RNQVlST Department of Physiological Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden
(First received May 1987; accepted in revised form February 1988) Abstract--Two green algae, Monoraphidium dybowskii and Stichococcus sp., were exposed to aluminium at some ecologically relevant concentrations within a pH range of 5.0-6.0. Growth experiments in batch cultures showed some difference in tolerance response between the two species. Cell decomposition was detectable at 0.10mgAll -I but was more pronounced at higher AI concentrations. Monoraphidium showed higher tolerance to aluminium than did Stichococcus when ECs0 values were considered. The growth rate of this alga was reduced to 50% of the control when it was exposed to 1 mg AI 1- t. The ECs0 value for Stichococcus was 0.52 mg AI 1-1. Upon measurement of the 50% reduction in maximum cell biomass, about the same values (0.4 mg A11-1) were noted for both algae. Calculation of the area under the growth curve gave a more integrated growth response resulting in intermediate values for both algae but still half as high for Stichococcus as for Monoraphidium. However, no effective concentration (NOEC) and least concentration significant effect (LCSE) of aluminium, were signified by lower values (around 0.02rag 1-I) for Monoraphidium compared with those (0.ll-0.23 mgl -I) for Stichococcus, which responded more dramatically when the AI concentration was increased. The increase in cell number production was progressively more affected during the growth experiments. Cells of Stichococcus, however, seemed to adapt to some extent after more than 3 days of exposure, showing higher tolerance at intermediate AI concentrations after some additional days of exposure.
Key words--alunfinium Stichococcus sp.
toxicity, green algae, dose response growth, Monoraphidium dybowskii,
INTRODUCTION
MATERIAL AND METHODS
Augmented acidification o f soil and water will change many biological processes and also influence the structure of the ecosystems. Rapid and dramatic alterations of the environmental conditions will drastically increase the stress on both living individuals and on the structure of biological communities. The tolerance against environmental stress and fluctuations differ greatly between, for example, different algal species. The number of species surviving with decreasing p H values in a water is not that great (Kwiatkowki and Roff, 1976; Almer et al., 1978; Johansson and Nyberg, 1981; Lyd6n and Grahn, 1985). When the p H values decrease, some metal ions become more soluble in the free water of a lake. One of the most interesting elements in this respect is aluminium, the third most c o m m o n in the crust of the earth. Increased amounts of aluminium have frequently been registered in acidified waters (Dickson, 1978; Wenblad and Johansson, 1980). It is, therefore, of great interest to investigate whether aluminium can be a selective factor for the composition of the surviving (resistant?) algal communities. A simple analysis of the aluminium resistance found a m o n g some different acido-tolerant algal species may, therefore, be the first step towards a deeper understanding of the environmental factors ruling growth (e.g. nutrients).
Two green algal species, Monoraphidium dybowskii (Wolosz.) Hind. and Kom.-Legn. and Stichococcus sp., were isolated from the slightly acidified Swedish lakes O. Nedsj6n (pH 5.5) and Sk~irvsj6n (pH 6.3), respectively. Both species were grown as stock cultures in Erlenmeyer flasks at 25°C and illuminated with continuous light (100 #E/m2s, PAR). Reinoculation once a week into a defined growth medium (10% L- 16; Lindstr6m, 1983) kept the culture exponentially growing. The concentration of phosphorus was, however, lowered to 5.6/ag 1-1 in the experiments, and 10 mM MES [2-(N-morpholino)-ethansulphonic acid] buffer was added to keep pH constant at 5.0, 5.5 or 6.0. The same composition of growth medium as for the stock cultures was also used in experiments to test the algal response to aluminium without EDTA. The light and temperature regimes were also kept the same. The concentration of aluminium, added as AICI3, followed a geometric dilution series (0, 0.10, 0.18, 0.32, 0.56, 1.00 and 1.80mg 1-~) prepared at the initiation of each experiment by using Millipore-active-carbon-filtered distilled water. Synthetic chelators were omitted in all culturing experiments. The algal cell suspension was diluted in each Erlenmeyer flask to an initial cell concentration of 10 7 cells 1-t (or 3 x 10 7 cells 1-' in the cell decomposition experiments). The cell number in each Erlenmeyer flask (normally three parallels were used) was determined by the use of a particle counter (Coulter Counter ZM) and the cell sizes, mean cell volume and total cell volume by the use of a C-1000 Channalyzer and a micro-computer (ABC-800). The cell cultures were all the time kept axenically growing. The minimum cell size of "living cells" was determined by studying the cell size distribution. The diserimination threshold between "living cells" and "decomposing cell 977
978
A. CLAESSOr~and L. TORNQVlST
matter" could be identified between two peaks in the size distribution (cf. T6mqvist and Claesson, 1987). This threshold was typically found around 7 #m 3 for Stichococcus but more undefined around 5 #m 3 for Monoraphidium. The term "cell decomposition" is used when particles smaller than normal algal cells were observed by the Channalyzer and verified microscopically. In cases when an extended lag phase in cell growth was observed this was regarded as a "delay in growth initiation" before the cell number started to increase. Growth rates were calculated from a best-fit (by eye) straight line in the exponential growth phase. The maximum biomass was established as the estimated maximum cell number producible, and areas under the growth curves were obtained by cumulative additions of areas between successive points of time (OECD, 1984). Cell number as well as log (cell number) was used. The significant differences between treatments were tested by the use of the ordinary t-test, and the dose-response calculations by the use of probit analysis. The concentration of a substance effective to reduce the growth rate to 50% of the control is called the ECs0-value of that substance.
pH-dependent growth Both algal species, Monoraphidium and Stichococcus, grew well at the investigated pH values of 5.0, 5.5 and 6.0. The growth curves looked similar in most respects, i.e. the lag growth phase, the exponential growth phase and the stationary growth phase. The growth rate values were typically around 1.1 day i for Monoraphidium at p H 5 . 5 and 6.0 but 1.3 day i at p H 5.0. Stichococcus grew a little slower, l.l day ~ at pH 5.0 and 5.5 and 1.0 day -~ at pH 6.0. Some more cells were, however, produced by Monoraphidium from the given amount of nutrients (cf. Figs 1 and 2). The total biomass expressed as cell volume was about 27 mm 3 1-~ for Monoraphidium compared with 12 m m 3 i -~ for Stichococcus resulting in the yield coefficients Y e = 4 . 9 m m 3 pg-~ P for
1000
o X U)
--
A 0f . . x
100-
(n
0
I
I
2
4
I
6
8
days
Fig. 2. Growth curves for Stichococcus sp. exposed to different concentrations of aluminium (mg I ]). Mean values of three parallels. 1 SD. Symbols as in Fig. 1.
Monoraphidium and Yp = 2.2 mm 3 # g J P for Stichococcus, assuming that phosphorus was the most
RESULTS
A
1000-
10o.
0
I
I
I
I
I
2
4
6
8
10
days
Fig. 1. Growth curves for Monoraphidium dybowskii exposed to different concentrations of aluminium (mg 1- ~). Mean values of three parallels. 1 SD C), control; A, 0.18; [], 0.32; O, 0.56; &, 1.00; l , 1.80.
limiting nutrient and completely exhausted by the algae. The N / P quotient of the medium was 38/1. The proportion between the maximum biomass of the two algal species have repeatedly been 2:1.
Al-dependent growth Exposure of the algae to aluminium resulted in different growth responses. Growth curves for Monoraphidium and Stichococcus at p H 5.0 are shown in Figs 1 and 2. Aluminium inhibited the growth of both algae. Delayed growth was found for Stichococcus, the higher the concentration of aluminium, the longer was the delay of growth initiation. For Monoraphidium an extended delay caused by aluminium was not as obvious as for Stichococcus.
Cell decomposition Some Stichococcus cells exposed to aluminium seemed to decompose, since the cell number decreased during the first 1 or 2 days after inoculation. In an attempt to calculate cell decomposition, rates about 0.36h J were found for cells exposed to 0.32 mg A! 1 ~, i.e. almost half the initial cell number was lost after 2.5 h, while only I/3 of that rate was found at 0.18 mg A1 1--~. N o decrease in cell number was noted for 0.10mg A I I -~ and for the control. With sole regard to particle sizes equal to or bigger than the initial inoculum cell size, the decrease in cell number was even more pronounced. The maximum decomposition rates were about the same for all AI concentrations above 0.18 mg 1- J, and the minimum cell number, expressed as per cent of the initial value, were 40, 28, 28 and 9 for the concentrations 0.18, 0.32, 0.56 and 1.00mg A I I -~, respectively. By employing mean cell size at each time of cell number assessment, a total cell volume was calculated. In Fig. 3 the change in this calculated total cell volume
Toxicity of A1 to acido-tolerant green algae
979
Stichococcus were normally used in the toxicity experiments. In order to get an indication of the AI influence on cell division, cells from the stationary growth phase were also inoculated into Al-gradient experiments. Some differences between the young cells ( = logarithmically growing) and the older cells ( = stationary growing) were found during the first 2 days after the start of the experiment. Young ( = dividing) cells decomposed within a few hours but the surviving ones started to divide during the first 32 h (Fig. 3). The older (non-dividing) cells did not start their decomposition as rapidly as the younger ones. However, cell decomposition occurred even at an A1 concentration of 0.10 mg 1-L but the growth of these cells was later stimulated (Fig. 4). The young cells were continuously stimulated in their growth in 0.10 mg AI 1-1. The start of cell division was detected after only 2.5h for the younger cells at 0.18mg A1 1-L but was delayed for about 22 h for the older cells. Older cells surviving 0.32mg A1 1-1 and 0.56mg A l l -~ started to divide after 2 or > 5 days, respectively. When cells derived from old ( = non-dividing) cells divided at their maximum rate ( = logarithmic growth phase), they showed a higher degree of aluminium sensitivity than those derived from young ( = dividing) cells. The ECs0 value in the latter case was about half the former.
400
100
15
30
45
TIME (HOt.S)
Fig. 3. Percentage change in total cell volume of logarithmicaUy growing Stichococcus cells exposed to aluminium (mg 1-~). O, control; V, 0.10; A, 0.18; I-q, 0.32; 0 , 0.56; &, 1.00.
can be followed during the first 2 days of growth. A rapid decrease in cell volume was found for cell samples exposed to AI concentrations of 0.32 mg I-l or higher. The "negative cell volume balance" was also detected at 0.18 mg AI 1-] during the first 9 h. However, at that AI concentration cells start to divide earlier than those exposed to higher concentrations. More than a 50% reduction in total cell volume was obtained when only "living cells" were considered (i.e. particles equal to or bigger than the cell size of the initial inoculum). The minimum total cell volumes were in the range of 70, 35, 35 and 10% of the initial values for the A1 concentrations of 0.18, 0.32, 0.56 and 1.00 mg 1-], respectively. The surviving cells started to divide after a delay of 2.5-32 h, the higher the AI concentration, the later the start. Even cells exposed to 1.00 mg AI 1-~ divided a few times. Monoraphidium did not show any sign of cell decomposition when studied in this way. On the other hand, investigations of the cell size distributions revealed changes in the population (cf. T6rnqvist and Claesson, 1987).
Young and older cells Cells from logarithmically growing cultures of
3OO
200
I
15
30
45
11ME (HOURS1
Fig. 4. Percentage change in total cell volume of stationarily growing Stichococcus cells exposed to aluminium (mg l-t). Symbols as in Fig. 3.
A. CLAESSONand L, TORNQVIST
980
detailed information can be obtained on the rate with oI m e=
° 80o 60.
.
.
40" t.,
"
.
.
.
.
fiSfi~,.o
~ M O N 5.5 MON6.0
20-
S1
o
\ ST1 5.0
Lm
0'.1 AI c o n c e n t r
1.0 a t i o n (rag/I)
Fig. 5. Growth rate responses (probit analysis) of Monoraphidium and Stichococcus exposed to aluminium at different pH levels (cf. data in Table 1).
Growth rate The growth rate of the algae was clearly influenced by additions of aluminium (cf. Figs 1 and 2). Growth rate values from three different experiments, each representing three parallels, yielded characteristic dose-response curves for the various pH values (Fig. 5). An aluminium concentration of 1.00 mg 1-1 limited the growth rate of Monoraphidium to 50% of the control at pH 5.0 and 5.5. At pH6.0 a 50% reduction was found for 0.55 mg AI 1-1. Stichococcus, however, showed quite another response to aluminium exposure. A much steeper change in growth was noted when the concentration of aluminium increased. The responses of Stichococcus were also quite different at the different pH values. These cells seemed to be most resistant at pH 5.0, exemplified by a 50% growth-rate reduction at 0.5 mg AI 1 ~. The corresponding value at pH 5.5 was around 0.22 mg A1 1--1. At pH 6.0 a similar dramatic change in algal growth rate was observed. The differences in ECs0 values found for the two algal species were even more pronounced when the "'no-effective concentrations" (NOEC) were calculated by means of probit analysis. Only the linear part of the original dose-response curve was employed (tested by chi2-analysis) in order to extrapolate the NOEC value. Stichococcus showed high NOEC values around 0.2 mg AI 1-I, while Monoraphidium responded to much lower A! concentrations. An almost complete growth inhibition of Stichococcus was induced by 1.00 mg A1 1-1 at pH-values 5.0 and 6.0. At pH 5.5 this effect was observed already at 0.32 mg A11 -]. A 100% inhibition of the growth rate was not noted for Monoraphidium (Fig. 5).
Cell production and maximum biomass Aiuminium also had an influence on the maximum biomass producible (Figs 1 and 2). By plotting the percentage of the cell-number responses at different points of time against the AI concentration more
which aluminium influences cell production. This was done for some selected time periods in Fig. 6. After a 5-day exposure relatively low concentrations of aluminium (0.32mg 1 l) gave rise to a significant reduction in cell number of Monoraphidium, while 1.00mg AI 1-1 or higher concentrations had an influence some days earlier. The decrease in cell number of Monoraphidium in relation to the control, continued as long as the growth experiment proceeded (cf. Fig. 1). The cell production of Stichococcus proceeded a little differently [Fig. 6(B)]. At the low AI concentration of 0.18 mg 1 i no real reduction was noted. However, higher concentrations of aluminium quickly decreased the cell number. Already after 0.1 days ( = 2,5 h) exposure to 1.00 mg A1 1-1, the cell number was reduced to 55% of the control, a decrease that continued during the whole experimental period. On the other hand, cells exposed to lower AI concentrations seemed to withstand the situation after some days of adaptation. This can be seen most clearly for cells treated with 0.32 mg AI 1-~, in which a 40% cell production after 3 days was doubled after 9 days of treatment.
Areas under growth curves Since aluminium can influence cell growth in different ways, e.g. through prolonged growth lag,
801A0 0.1
.... 1.0
B 8060-
0.1
402
20I
I
0.1
1.0
AI-r..~NCENq~4ATION (~/l)
Fig. 6. Cell number of Monoraphidium (A) and Stichococcus (B) expressed as % of control after exposure to aluminium (mg l -I ) during different time periods (days). Mean values of three parallels.
Toxicity of A1 to acido-tolerant green algae
981
Table 1. Values for no effective concentration ( N O E C ) , least concentration significant effect (LCSE), effective concentration to a 50% reduction (ECs0) with 9 5 % confidence limits and values o f the slope (k) for dose-response lines for three different algal growth parameters at p H 5.0. D a t a in m g AI 1-1
Monoraphidium Growth parameter G r o w t h rate M a x i m u m cell biomass A r e a under growth curve
Stichococcus
NOEC
LCSE
ECso
k
NOEC
LCSE
ECso
k
0.04 0.02 0.02
0.11 0.06 0.12
I.l I _+ 0.07 0.40 _+ 0.04 0.78 __ 0.10
-46.7 - 131 -66.0
0,23 0.11 0.12
0.24 0,14 0.14
0.52 _+ 0.01 0.42 +_ 0.02 0.32 __ 0.01
- 172 - 160 -250
cell decomposition, inhibited growth rate and decreased cell biomass, a method suggested to integrate all of these growth parameters was tested. The total area under the different growth curves was calculated for each AI concentration and was compared with the area of the control curve. Some dose-response data are thus presented in Table 1.
Comparison of growth-response parameters The degree of inhibition caused by aluminium was compared at pH 5.0 for the two algae as well as their response parameters growth rate, maximum cell biomass and area under growth curves (Table 1). The no-effect concentration (NOEC) varied by a factor of two between the three growth parameters, at which the growth rates showed the highest values. The lowest concentration with significant effect (LCSE) was assessed as the upper 95% confidence limit of the NOEC value. These values also differed by a factor of two. The ECs0 values obtained through probit analysis (including only data with a good fit along a straight dose-response line. i.e. an exclusion of t h e data representing the lower A1 concentrations) were somewhat more scattered. For Monoraphidium the highest value for growth rate was 1.11 mg AI 1- J and the lowest 0.40 mg A1 I-l, both obtained when maximal cell biomass was estimated. The growth rate of Stichococcus also corresponded to the highest ECs0 value (0.52 mg A1 I-l), while the lowest ECho value (0.32 mg AI 1-~) was obtained when the area under the growth curve was used as a growth parameter. All EC~0 values were significantly different from each other. The calculation of the area under the growth curve by means of log (cell number) did not offer any further advantages in these cases. The ECs0 values calculated by means of maximum cell biomass were
about the same for the two algae, while the ECs0 values for growth rate and area under growth curve were about half as high for Stichococcus as for Monoraphidium. The linear slope of the doseresponse relationship gives information about the decrease of the algal growth with increasing AI concentrations. The values for Stichococcus were closer to each other than they were for Monoraphidium. The slope of the curve for maximum cell biomass of Monoraphidium was the steepest and almost similar to the ones of Stichococcus. The steepest slope was found for Stichococcus when the area under the growth curve was used. An analysis of variance makes it possible to perceive if a treatment of aluminium gives a different response of algal growth to that of the control (Table 2). The algae responded accordingly with great significance to the higher A1 concentrations irrespective of any one of the different growth parameters. At 0.32 mg A11 -~ or lower concentrations, Stichococcus could not be found to respond unless the area under the growth curve was calculated. The response of Monoraphidium at these concentrations was indicative only if growth rate was measured. DISCUSSION
Monoraphidium and Stichococcus were selected as test organisms, since both grew well at relatively low pH values. The number of life forms (e.g. different algal species) is reduced in acidified lakes. This reduction seems to be especially pronounced at pH values of 5.0-6.0 (see e.g. Lyd6n and Grahn, 1985). In this range aluminium mainly exists in the form of Al 3+, AI(OH) 2+ and AI(OH)~. HelliweU et al. (1983) found Al(OH)f to be most toxic of Chlorella pyrenoidosa.
Table 2. Probability o f a treatment resulting in a response different to the control. N S = not significant, * = significant at 9 5 % level, * * = significant at 9 9 % level, *** = significant at 99.9% level Al-conc. ( m g 1 ~)
M a x i m u m cell number
0.18 0.32 0.56 1.00
NS NS * **
0.18 0.32 0.56 1.00
NS NS ** ***
Growth rate
A r e a under growth curve
L o g area under growth curve
NS * * ***
NS NS ** ***
* * *** ***
*** *** *** ***
Monoraphidium ** ** *** ***
Stichococcus NS NS ** ***
982
A. CLAF.SSONand L. TORNQVlST
In the present study aluminium was found to inhibit growth of Monoraphidium and Stichococcus to different degrees. A delayed growth initiation was observed for Stichococcus. This temporary inhibition of cell division may be due to initial death and decomposition of some cells susceptible to aluminium, or physiological adaptation of the cells to aluminium after some time. This adaptation may have its origin in different metabolic modifications in the cell making it possible to start its growth at some later moment. Abiotic changes of the ambient chemistry can also, after some time, give rise to non-toxic levels of aluminium for the algal cells. A decomposition of Stichococcus cells did really occur, since a dramatic decrease in cell number was observed already after a few hours of AI exposure. The surviving cells started to divide after various time periods. Cells treated with the highest AI concentration showed the longest delay. Older cells (from stationary growth phase) were more resistant to aluminium with regard to immediate cell decomposition. This indicates that an influence of aluminium is being exerted on the activity of cell division. However, the daughter cells of cells exposed to aluminium during non-dividing conditions showed a reduced dividing capacity. Whether the young or the older cells are most sensitive to aluminium is further discussed elsewhere by T6rnqvist and Claesson (1987). In an attempt to detect the inhibitory effects of the aluminium ion on cell growth and also possibly discriminate between the two algal species different growth parameters were studied. Monoraphidium seemed rather insensitive at a first quick glance except for a low reduction in growth rate and maximum cell. biomass production. Although this growth parameter was paralleled by the highest ECs0 value, it was the most sensitive as an indicator of cell response to aluminium compared to that of the control. Growth inhibition of Stichococcus was detected most easily by studying the area under the growth curve. Comparison of the growth responses for the two green algae revealed some interesting differences. As already mentioned above, some Stichococcus cells decomposed more or less immediately upon exposure to aluminium. This could not be found for Monoraphidium, and the obvious delay in initiation of cell division shown by Stichococcus was not found for Monoraphidium. However, the growth rate was reduced at much lower AI concentrations for Monoraphidium even though the ECs0 value was about twice as high as that for Stichococcus. This observation was valid for all observed growth parameters with the exception that the ECs0 values for maximum cell biomass were about the same. Even though Monoraphidium was more sensitive to low AI concentrations, this alga tolerated higher concentrations better than Stichococcus. The latter showed a dramatic growth response, when the A1 concentration
in the medium was doubled from NOEC levels. Aluminium may thus affect the two algae in much different ways. Stichococcus cells exposed to moderate AI concentrations also seemed to increase their tolerance after some days of exposure. This phenomenon will be further investigated. The lowest concentration of aluminium that significantly affected algal cell growth was in the range of 0.6--0.24 mg 1-~ at pH 5.0 (Table 1). These values correspond very well with those found in lakes from the Swedish west coast region (Dickson, 1978). Our two green algal species may then subsist at their "ecological borderline", regarding in situ AI concentrations, although they can accept lower pH values than those measured in the lake waters when they were isolated. Stichococcus will probably be the first "looser" if the AI concentration increases in the lakes, since this alga so dramatically responded to the higher AI concentration levels. Upon scrutiny of the NOEC values instead of the LCSE values (Table 1) we find Monoraphidium to be even more sensitive to aluminium. This can be explained by the greater variance in the acquired data for Monoraphidium. NOEC values estimated through a best fit (by eye) line without any probit analysis gave somewhat higher values for both algae. This was partly caused by the growth stimulatory effect of low AI concentrations. H6rnstrrm et al. (1984) reported Monoraphidium dybowskii to be "uneffected by 400 #g AI I I as most crysophyceans do (p. 121)". They measured cell number fluorimetrically as well as by visual cell inspection after 3-4 days of growth. However, our results suggest Monoraphidium to be more sensitive to aluminium, which is most clearly indicated by complete growth curves. Maximum cell biomass was found to be the most sensitive growth parameter for this alga. H6rnstr6m et al. (1984) also noted very small concentration differences between growth reduction and inhibition for desmids and diatoms in the AI concentration range of 100-150 #g AI 1- J. We recorded such a marked difference also for our two investigated green algae, but at some higher AI concentrations. Acknowledgements--We thank Dr E. Brammer for valuable comments and for linguistic revision of the manuscript. This study was partly supported by grants from Lennanders and Wallins fund at the University of Uppsala, Sweden and also from the Hierta-Retzius fund at the Royal Swedish Academy of Science.
REFERENCES
Almer B., Dickson W., Ekstr6m C. and H6mstr6m E. (1978) Sulphur pollution and the aquatic ecosystem. In Sulphur in the Environment, Part H (Edited by Nriagu I. O.), pp. 271-286. Dickson W. (1978) Some effects of the acidification of Swedish lakes. Verh. int. Verein. theor, angew. Limnol. 20, 851-856. Helliwell S., Battey G. E., Florence T. M. and Lumsden
Toxicity of AI to acido-tolerant green algae B. G. (1983) Speciation and toxicity of aluminium in a model freshwater. Envir. Technol. Lett. 4 No. 3, 141-144. H6rnstr6m E., Ekstr6m C. and Duraini O. (1984) Effects of pH and different levels of aluminium on lake plankton in the Swedish west coast area. Natn. Swed. Bd Fish. Report No. 61. Johansson K. and Nyberg P. (1981) F6rsurning av svenska ytvatten-effekter och omfattning 1980. Information frdn s6tvattenslab. No. 6. (English summary). Kwiatkowski R. E. and Roff J. C. (1976) Effects of acidity on the phytoplankton and primary productivity of selected northern Ontario lakes. Can. J. Bot. 54, 2546-2561. Lindstr6m K. (1983) Selenium as a growth factor for
983
plankton algae in laboratory experiments and in some Swedish lakes. Hydrobiologia 101, 35-48. Lyd6n A. and Grahn O. (1985) Phytoplankton species composition, biomass and production in Lake G~rdsj6n--an acidified clearwater lake in SW Sweden. Ecol. Bull. (Stockholm) 37, 195-202. OECD (1984) Guideline for Testing of Chemicals. Alga, Growth inhibition Test, p. 210. OECD, Paris. T6rnqvist L. and Claesson A. (1987) The influence of aluminium on the cell-size distribution of two green algae. Envir. expl Bot. 27, 481-488. Wenblad A. and Johansson A. (1980) Aluminium i f6rsurade v/istsvenska sj6ar. Vatten 36, 154-157 (English summary).
0043-1354/88 $3.00+0.00 Copyright © 1988 Pergamon Press plc
THE TOXICITY OF ALUMINIUM TO TWO ACIDO-TOLERANT GREEN ALGAE A. CLAESSON and L. T6RNQVlST Department of Physiological Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden
(First received May 1987; accepted in revised form February 1988) Abstract--Two green algae, Monoraphidium dybowskii and Stichococcus sp., were exposed to aluminium at some ecologically relevant concentrations within a pH range of 5.0-6.0. Growth experiments in batch cultures showed some difference in tolerance response between the two species. Cell decomposition was detectable at 0.10mgAll -I but was more pronounced at higher AI concentrations. Monoraphidium showed higher tolerance to aluminium than did Stichococcus when ECs0 values were considered. The growth rate of this alga was reduced to 50% of the control when it was exposed to 1 mg AI 1- t. The ECs0 value for Stichococcus was 0.52 mg AI 1-1. Upon measurement of the 50% reduction in maximum cell biomass, about the same values (0.4 mg A11-1) were noted for both algae. Calculation of the area under the growth curve gave a more integrated growth response resulting in intermediate values for both algae but still half as high for Stichococcus as for Monoraphidium. However, no effective concentration (NOEC) and least concentration significant effect (LCSE) of aluminium, were signified by lower values (around 0.02rag 1-I) for Monoraphidium compared with those (0.ll-0.23 mgl -I) for Stichococcus, which responded more dramatically when the AI concentration was increased. The increase in cell number production was progressively more affected during the growth experiments. Cells of Stichococcus, however, seemed to adapt to some extent after more than 3 days of exposure, showing higher tolerance at intermediate AI concentrations after some additional days of exposure.
Key words--alunfinium Stichococcus sp.
toxicity, green algae, dose response growth, Monoraphidium dybowskii,
INTRODUCTION
MATERIAL AND METHODS
Augmented acidification o f soil and water will change many biological processes and also influence the structure of the ecosystems. Rapid and dramatic alterations of the environmental conditions will drastically increase the stress on both living individuals and on the structure of biological communities. The tolerance against environmental stress and fluctuations differ greatly between, for example, different algal species. The number of species surviving with decreasing p H values in a water is not that great (Kwiatkowki and Roff, 1976; Almer et al., 1978; Johansson and Nyberg, 1981; Lyd6n and Grahn, 1985). When the p H values decrease, some metal ions become more soluble in the free water of a lake. One of the most interesting elements in this respect is aluminium, the third most c o m m o n in the crust of the earth. Increased amounts of aluminium have frequently been registered in acidified waters (Dickson, 1978; Wenblad and Johansson, 1980). It is, therefore, of great interest to investigate whether aluminium can be a selective factor for the composition of the surviving (resistant?) algal communities. A simple analysis of the aluminium resistance found a m o n g some different acido-tolerant algal species may, therefore, be the first step towards a deeper understanding of the environmental factors ruling growth (e.g. nutrients).
Two green algal species, Monoraphidium dybowskii (Wolosz.) Hind. and Kom.-Legn. and Stichococcus sp., were isolated from the slightly acidified Swedish lakes O. Nedsj6n (pH 5.5) and Sk~irvsj6n (pH 6.3), respectively. Both species were grown as stock cultures in Erlenmeyer flasks at 25°C and illuminated with continuous light (100 #E/m2s, PAR). Reinoculation once a week into a defined growth medium (10% L- 16; Lindstr6m, 1983) kept the culture exponentially growing. The concentration of phosphorus was, however, lowered to 5.6/ag 1-1 in the experiments, and 10 mM MES [2-(N-morpholino)-ethansulphonic acid] buffer was added to keep pH constant at 5.0, 5.5 or 6.0. The same composition of growth medium as for the stock cultures was also used in experiments to test the algal response to aluminium without EDTA. The light and temperature regimes were also kept the same. The concentration of aluminium, added as AICI3, followed a geometric dilution series (0, 0.10, 0.18, 0.32, 0.56, 1.00 and 1.80mg 1-~) prepared at the initiation of each experiment by using Millipore-active-carbon-filtered distilled water. Synthetic chelators were omitted in all culturing experiments. The algal cell suspension was diluted in each Erlenmeyer flask to an initial cell concentration of 10 7 cells 1-t (or 3 x 10 7 cells 1-' in the cell decomposition experiments). The cell number in each Erlenmeyer flask (normally three parallels were used) was determined by the use of a particle counter (Coulter Counter ZM) and the cell sizes, mean cell volume and total cell volume by the use of a C-1000 Channalyzer and a micro-computer (ABC-800). The cell cultures were all the time kept axenically growing. The minimum cell size of "living cells" was determined by studying the cell size distribution. The diserimination threshold between "living cells" and "decomposing cell 977
978
A. CLAESSOr~and L. TORNQVlST
matter" could be identified between two peaks in the size distribution (cf. T6mqvist and Claesson, 1987). This threshold was typically found around 7 #m 3 for Stichococcus but more undefined around 5 #m 3 for Monoraphidium. The term "cell decomposition" is used when particles smaller than normal algal cells were observed by the Channalyzer and verified microscopically. In cases when an extended lag phase in cell growth was observed this was regarded as a "delay in growth initiation" before the cell number started to increase. Growth rates were calculated from a best-fit (by eye) straight line in the exponential growth phase. The maximum biomass was established as the estimated maximum cell number producible, and areas under the growth curves were obtained by cumulative additions of areas between successive points of time (OECD, 1984). Cell number as well as log (cell number) was used. The significant differences between treatments were tested by the use of the ordinary t-test, and the dose-response calculations by the use of probit analysis. The concentration of a substance effective to reduce the growth rate to 50% of the control is called the ECs0-value of that substance.
pH-dependent growth Both algal species, Monoraphidium and Stichococcus, grew well at the investigated pH values of 5.0, 5.5 and 6.0. The growth curves looked similar in most respects, i.e. the lag growth phase, the exponential growth phase and the stationary growth phase. The growth rate values were typically around 1.1 day i for Monoraphidium at p H 5 . 5 and 6.0 but 1.3 day i at p H 5.0. Stichococcus grew a little slower, l.l day ~ at pH 5.0 and 5.5 and 1.0 day -~ at pH 6.0. Some more cells were, however, produced by Monoraphidium from the given amount of nutrients (cf. Figs 1 and 2). The total biomass expressed as cell volume was about 27 mm 3 1-~ for Monoraphidium compared with 12 m m 3 i -~ for Stichococcus resulting in the yield coefficients Y e = 4 . 9 m m 3 pg-~ P for
1000
o X U)
--
A 0f . . x
100-
(n
0
I
I
2
4
I
6
8
days
Fig. 2. Growth curves for Stichococcus sp. exposed to different concentrations of aluminium (mg I ]). Mean values of three parallels. 1 SD. Symbols as in Fig. 1.
Monoraphidium and Yp = 2.2 mm 3 # g J P for Stichococcus, assuming that phosphorus was the most
RESULTS
A
1000-
10o.
0
I
I
I
I
I
2
4
6
8
10
days
Fig. 1. Growth curves for Monoraphidium dybowskii exposed to different concentrations of aluminium (mg 1- ~). Mean values of three parallels. 1 SD C), control; A, 0.18; [], 0.32; O, 0.56; &, 1.00; l , 1.80.
limiting nutrient and completely exhausted by the algae. The N / P quotient of the medium was 38/1. The proportion between the maximum biomass of the two algal species have repeatedly been 2:1.
Al-dependent growth Exposure of the algae to aluminium resulted in different growth responses. Growth curves for Monoraphidium and Stichococcus at p H 5.0 are shown in Figs 1 and 2. Aluminium inhibited the growth of both algae. Delayed growth was found for Stichococcus, the higher the concentration of aluminium, the longer was the delay of growth initiation. For Monoraphidium an extended delay caused by aluminium was not as obvious as for Stichococcus.
Cell decomposition Some Stichococcus cells exposed to aluminium seemed to decompose, since the cell number decreased during the first 1 or 2 days after inoculation. In an attempt to calculate cell decomposition, rates about 0.36h J were found for cells exposed to 0.32 mg A! 1 ~, i.e. almost half the initial cell number was lost after 2.5 h, while only I/3 of that rate was found at 0.18 mg A1 1--~. N o decrease in cell number was noted for 0.10mg A I I -~ and for the control. With sole regard to particle sizes equal to or bigger than the initial inoculum cell size, the decrease in cell number was even more pronounced. The maximum decomposition rates were about the same for all AI concentrations above 0.18 mg 1- J, and the minimum cell number, expressed as per cent of the initial value, were 40, 28, 28 and 9 for the concentrations 0.18, 0.32, 0.56 and 1.00mg A I I -~, respectively. By employing mean cell size at each time of cell number assessment, a total cell volume was calculated. In Fig. 3 the change in this calculated total cell volume
Toxicity of A1 to acido-tolerant green algae
979
Stichococcus were normally used in the toxicity experiments. In order to get an indication of the AI influence on cell division, cells from the stationary growth phase were also inoculated into Al-gradient experiments. Some differences between the young cells ( = logarithmically growing) and the older cells ( = stationary growing) were found during the first 2 days after the start of the experiment. Young ( = dividing) cells decomposed within a few hours but the surviving ones started to divide during the first 32 h (Fig. 3). The older (non-dividing) cells did not start their decomposition as rapidly as the younger ones. However, cell decomposition occurred even at an A1 concentration of 0.10 mg 1-L but the growth of these cells was later stimulated (Fig. 4). The young cells were continuously stimulated in their growth in 0.10 mg AI 1-1. The start of cell division was detected after only 2.5h for the younger cells at 0.18mg A1 1-L but was delayed for about 22 h for the older cells. Older cells surviving 0.32mg A1 1-1 and 0.56mg A l l -~ started to divide after 2 or > 5 days, respectively. When cells derived from old ( = non-dividing) cells divided at their maximum rate ( = logarithmic growth phase), they showed a higher degree of aluminium sensitivity than those derived from young ( = dividing) cells. The ECs0 value in the latter case was about half the former.
400
100
15
30
45
TIME (HOt.S)
Fig. 3. Percentage change in total cell volume of logarithmicaUy growing Stichococcus cells exposed to aluminium (mg 1-~). O, control; V, 0.10; A, 0.18; I-q, 0.32; 0 , 0.56; &, 1.00.
can be followed during the first 2 days of growth. A rapid decrease in cell volume was found for cell samples exposed to AI concentrations of 0.32 mg I-l or higher. The "negative cell volume balance" was also detected at 0.18 mg AI 1-] during the first 9 h. However, at that AI concentration cells start to divide earlier than those exposed to higher concentrations. More than a 50% reduction in total cell volume was obtained when only "living cells" were considered (i.e. particles equal to or bigger than the cell size of the initial inoculum). The minimum total cell volumes were in the range of 70, 35, 35 and 10% of the initial values for the A1 concentrations of 0.18, 0.32, 0.56 and 1.00 mg 1-], respectively. The surviving cells started to divide after a delay of 2.5-32 h, the higher the AI concentration, the later the start. Even cells exposed to 1.00 mg AI 1-~ divided a few times. Monoraphidium did not show any sign of cell decomposition when studied in this way. On the other hand, investigations of the cell size distributions revealed changes in the population (cf. T6rnqvist and Claesson, 1987).
Young and older cells Cells from logarithmically growing cultures of
3OO
200
I
15
30
45
11ME (HOURS1
Fig. 4. Percentage change in total cell volume of stationarily growing Stichococcus cells exposed to aluminium (mg l-t). Symbols as in Fig. 3.
A. CLAESSONand L, TORNQVIST
980
detailed information can be obtained on the rate with oI m e=
° 80o 60.
.
.
40" t.,
"
.
.
.
.
fiSfi~,.o
~ M O N 5.5 MON6.0
20-
S1
o
\ ST1 5.0
Lm
0'.1 AI c o n c e n t r
1.0 a t i o n (rag/I)
Fig. 5. Growth rate responses (probit analysis) of Monoraphidium and Stichococcus exposed to aluminium at different pH levels (cf. data in Table 1).
Growth rate The growth rate of the algae was clearly influenced by additions of aluminium (cf. Figs 1 and 2). Growth rate values from three different experiments, each representing three parallels, yielded characteristic dose-response curves for the various pH values (Fig. 5). An aluminium concentration of 1.00 mg 1-1 limited the growth rate of Monoraphidium to 50% of the control at pH 5.0 and 5.5. At pH6.0 a 50% reduction was found for 0.55 mg AI 1-1. Stichococcus, however, showed quite another response to aluminium exposure. A much steeper change in growth was noted when the concentration of aluminium increased. The responses of Stichococcus were also quite different at the different pH values. These cells seemed to be most resistant at pH 5.0, exemplified by a 50% growth-rate reduction at 0.5 mg AI 1 ~. The corresponding value at pH 5.5 was around 0.22 mg A1 1--1. At pH 6.0 a similar dramatic change in algal growth rate was observed. The differences in ECs0 values found for the two algal species were even more pronounced when the "'no-effective concentrations" (NOEC) were calculated by means of probit analysis. Only the linear part of the original dose-response curve was employed (tested by chi2-analysis) in order to extrapolate the NOEC value. Stichococcus showed high NOEC values around 0.2 mg AI 1-I, while Monoraphidium responded to much lower A! concentrations. An almost complete growth inhibition of Stichococcus was induced by 1.00 mg A1 1-1 at pH-values 5.0 and 6.0. At pH 5.5 this effect was observed already at 0.32 mg A11 -]. A 100% inhibition of the growth rate was not noted for Monoraphidium (Fig. 5).
Cell production and maximum biomass Aiuminium also had an influence on the maximum biomass producible (Figs 1 and 2). By plotting the percentage of the cell-number responses at different points of time against the AI concentration more
which aluminium influences cell production. This was done for some selected time periods in Fig. 6. After a 5-day exposure relatively low concentrations of aluminium (0.32mg 1 l) gave rise to a significant reduction in cell number of Monoraphidium, while 1.00mg AI 1-1 or higher concentrations had an influence some days earlier. The decrease in cell number of Monoraphidium in relation to the control, continued as long as the growth experiment proceeded (cf. Fig. 1). The cell production of Stichococcus proceeded a little differently [Fig. 6(B)]. At the low AI concentration of 0.18 mg 1 i no real reduction was noted. However, higher concentrations of aluminium quickly decreased the cell number. Already after 0.1 days ( = 2,5 h) exposure to 1.00 mg A1 1-1, the cell number was reduced to 55% of the control, a decrease that continued during the whole experimental period. On the other hand, cells exposed to lower AI concentrations seemed to withstand the situation after some days of adaptation. This can be seen most clearly for cells treated with 0.32 mg AI 1-~, in which a 40% cell production after 3 days was doubled after 9 days of treatment.
Areas under growth curves Since aluminium can influence cell growth in different ways, e.g. through prolonged growth lag,
801A0 0.1
.... 1.0
B 8060-
0.1
402
20I
I
0.1
1.0
AI-r..~NCENq~4ATION (~/l)
Fig. 6. Cell number of Monoraphidium (A) and Stichococcus (B) expressed as % of control after exposure to aluminium (mg l -I ) during different time periods (days). Mean values of three parallels.
Toxicity of A1 to acido-tolerant green algae
981
Table 1. Values for no effective concentration ( N O E C ) , least concentration significant effect (LCSE), effective concentration to a 50% reduction (ECs0) with 9 5 % confidence limits and values o f the slope (k) for dose-response lines for three different algal growth parameters at p H 5.0. D a t a in m g AI 1-1
Monoraphidium Growth parameter G r o w t h rate M a x i m u m cell biomass A r e a under growth curve
Stichococcus
NOEC
LCSE
ECso
k
NOEC
LCSE
ECso
k
0.04 0.02 0.02
0.11 0.06 0.12
I.l I _+ 0.07 0.40 _+ 0.04 0.78 __ 0.10
-46.7 - 131 -66.0
0,23 0.11 0.12
0.24 0,14 0.14
0.52 _+ 0.01 0.42 +_ 0.02 0.32 __ 0.01
- 172 - 160 -250
cell decomposition, inhibited growth rate and decreased cell biomass, a method suggested to integrate all of these growth parameters was tested. The total area under the different growth curves was calculated for each AI concentration and was compared with the area of the control curve. Some dose-response data are thus presented in Table 1.
Comparison of growth-response parameters The degree of inhibition caused by aluminium was compared at pH 5.0 for the two algae as well as their response parameters growth rate, maximum cell biomass and area under growth curves (Table 1). The no-effect concentration (NOEC) varied by a factor of two between the three growth parameters, at which the growth rates showed the highest values. The lowest concentration with significant effect (LCSE) was assessed as the upper 95% confidence limit of the NOEC value. These values also differed by a factor of two. The ECs0 values obtained through probit analysis (including only data with a good fit along a straight dose-response line. i.e. an exclusion of t h e data representing the lower A1 concentrations) were somewhat more scattered. For Monoraphidium the highest value for growth rate was 1.11 mg AI 1- J and the lowest 0.40 mg A1 I-l, both obtained when maximal cell biomass was estimated. The growth rate of Stichococcus also corresponded to the highest ECs0 value (0.52 mg A1 I-l), while the lowest ECho value (0.32 mg AI 1-~) was obtained when the area under the growth curve was used as a growth parameter. All EC~0 values were significantly different from each other. The calculation of the area under the growth curve by means of log (cell number) did not offer any further advantages in these cases. The ECs0 values calculated by means of maximum cell biomass were
about the same for the two algae, while the ECs0 values for growth rate and area under growth curve were about half as high for Stichococcus as for Monoraphidium. The linear slope of the doseresponse relationship gives information about the decrease of the algal growth with increasing AI concentrations. The values for Stichococcus were closer to each other than they were for Monoraphidium. The slope of the curve for maximum cell biomass of Monoraphidium was the steepest and almost similar to the ones of Stichococcus. The steepest slope was found for Stichococcus when the area under the growth curve was used. An analysis of variance makes it possible to perceive if a treatment of aluminium gives a different response of algal growth to that of the control (Table 2). The algae responded accordingly with great significance to the higher A1 concentrations irrespective of any one of the different growth parameters. At 0.32 mg A11 -~ or lower concentrations, Stichococcus could not be found to respond unless the area under the growth curve was calculated. The response of Monoraphidium at these concentrations was indicative only if growth rate was measured. DISCUSSION
Monoraphidium and Stichococcus were selected as test organisms, since both grew well at relatively low pH values. The number of life forms (e.g. different algal species) is reduced in acidified lakes. This reduction seems to be especially pronounced at pH values of 5.0-6.0 (see e.g. Lyd6n and Grahn, 1985). In this range aluminium mainly exists in the form of Al 3+, AI(OH) 2+ and AI(OH)~. HelliweU et al. (1983) found Al(OH)f to be most toxic of Chlorella pyrenoidosa.
Table 2. Probability o f a treatment resulting in a response different to the control. N S = not significant, * = significant at 9 5 % level, * * = significant at 9 9 % level, *** = significant at 99.9% level Al-conc. ( m g 1 ~)
M a x i m u m cell number
0.18 0.32 0.56 1.00
NS NS * **
0.18 0.32 0.56 1.00
NS NS ** ***
Growth rate
A r e a under growth curve
L o g area under growth curve
NS * * ***
NS NS ** ***
* * *** ***
*** *** *** ***
Monoraphidium ** ** *** ***
Stichococcus NS NS ** ***
982
A. CLAF.SSONand L. TORNQVlST
In the present study aluminium was found to inhibit growth of Monoraphidium and Stichococcus to different degrees. A delayed growth initiation was observed for Stichococcus. This temporary inhibition of cell division may be due to initial death and decomposition of some cells susceptible to aluminium, or physiological adaptation of the cells to aluminium after some time. This adaptation may have its origin in different metabolic modifications in the cell making it possible to start its growth at some later moment. Abiotic changes of the ambient chemistry can also, after some time, give rise to non-toxic levels of aluminium for the algal cells. A decomposition of Stichococcus cells did really occur, since a dramatic decrease in cell number was observed already after a few hours of AI exposure. The surviving cells started to divide after various time periods. Cells treated with the highest AI concentration showed the longest delay. Older cells (from stationary growth phase) were more resistant to aluminium with regard to immediate cell decomposition. This indicates that an influence of aluminium is being exerted on the activity of cell division. However, the daughter cells of cells exposed to aluminium during non-dividing conditions showed a reduced dividing capacity. Whether the young or the older cells are most sensitive to aluminium is further discussed elsewhere by T6rnqvist and Claesson (1987). In an attempt to detect the inhibitory effects of the aluminium ion on cell growth and also possibly discriminate between the two algal species different growth parameters were studied. Monoraphidium seemed rather insensitive at a first quick glance except for a low reduction in growth rate and maximum cell. biomass production. Although this growth parameter was paralleled by the highest ECs0 value, it was the most sensitive as an indicator of cell response to aluminium compared to that of the control. Growth inhibition of Stichococcus was detected most easily by studying the area under the growth curve. Comparison of the growth responses for the two green algae revealed some interesting differences. As already mentioned above, some Stichococcus cells decomposed more or less immediately upon exposure to aluminium. This could not be found for Monoraphidium, and the obvious delay in initiation of cell division shown by Stichococcus was not found for Monoraphidium. However, the growth rate was reduced at much lower AI concentrations for Monoraphidium even though the ECs0 value was about twice as high as that for Stichococcus. This observation was valid for all observed growth parameters with the exception that the ECs0 values for maximum cell biomass were about the same. Even though Monoraphidium was more sensitive to low AI concentrations, this alga tolerated higher concentrations better than Stichococcus. The latter showed a dramatic growth response, when the A1 concentration
in the medium was doubled from NOEC levels. Aluminium may thus affect the two algae in much different ways. Stichococcus cells exposed to moderate AI concentrations also seemed to increase their tolerance after some days of exposure. This phenomenon will be further investigated. The lowest concentration of aluminium that significantly affected algal cell growth was in the range of 0.6--0.24 mg 1-~ at pH 5.0 (Table 1). These values correspond very well with those found in lakes from the Swedish west coast region (Dickson, 1978). Our two green algal species may then subsist at their "ecological borderline", regarding in situ AI concentrations, although they can accept lower pH values than those measured in the lake waters when they were isolated. Stichococcus will probably be the first "looser" if the AI concentration increases in the lakes, since this alga so dramatically responded to the higher AI concentration levels. Upon scrutiny of the NOEC values instead of the LCSE values (Table 1) we find Monoraphidium to be even more sensitive to aluminium. This can be explained by the greater variance in the acquired data for Monoraphidium. NOEC values estimated through a best fit (by eye) line without any probit analysis gave somewhat higher values for both algae. This was partly caused by the growth stimulatory effect of low AI concentrations. H6rnstrrm et al. (1984) reported Monoraphidium dybowskii to be "uneffected by 400 #g AI I I as most crysophyceans do (p. 121)". They measured cell number fluorimetrically as well as by visual cell inspection after 3-4 days of growth. However, our results suggest Monoraphidium to be more sensitive to aluminium, which is most clearly indicated by complete growth curves. Maximum cell biomass was found to be the most sensitive growth parameter for this alga. H6rnstr6m et al. (1984) also noted very small concentration differences between growth reduction and inhibition for desmids and diatoms in the AI concentration range of 100-150 #g AI 1- J. We recorded such a marked difference also for our two investigated green algae, but at some higher AI concentrations. Acknowledgements--We thank Dr E. Brammer for valuable comments and for linguistic revision of the manuscript. This study was partly supported by grants from Lennanders and Wallins fund at the University of Uppsala, Sweden and also from the Hierta-Retzius fund at the Royal Swedish Academy of Science.
REFERENCES
Almer B., Dickson W., Ekstr6m C. and H6mstr6m E. (1978) Sulphur pollution and the aquatic ecosystem. In Sulphur in the Environment, Part H (Edited by Nriagu I. O.), pp. 271-286. Dickson W. (1978) Some effects of the acidification of Swedish lakes. Verh. int. Verein. theor, angew. Limnol. 20, 851-856. Helliwell S., Battey G. E., Florence T. M. and Lumsden
Toxicity of AI to acido-tolerant green algae B. G. (1983) Speciation and toxicity of aluminium in a model freshwater. Envir. Technol. Lett. 4 No. 3, 141-144. H6rnstr6m E., Ekstr6m C. and Duraini O. (1984) Effects of pH and different levels of aluminium on lake plankton in the Swedish west coast area. Natn. Swed. Bd Fish. Report No. 61. Johansson K. and Nyberg P. (1981) F6rsurning av svenska ytvatten-effekter och omfattning 1980. Information frdn s6tvattenslab. No. 6. (English summary). Kwiatkowski R. E. and Roff J. C. (1976) Effects of acidity on the phytoplankton and primary productivity of selected northern Ontario lakes. Can. J. Bot. 54, 2546-2561. Lindstr6m K. (1983) Selenium as a growth factor for
983
plankton algae in laboratory experiments and in some Swedish lakes. Hydrobiologia 101, 35-48. Lyd6n A. and Grahn O. (1985) Phytoplankton species composition, biomass and production in Lake G~rdsj6n--an acidified clearwater lake in SW Sweden. Ecol. Bull. (Stockholm) 37, 195-202. OECD (1984) Guideline for Testing of Chemicals. Alga, Growth inhibition Test, p. 210. OECD, Paris. T6rnqvist L. and Claesson A. (1987) The influence of aluminium on the cell-size distribution of two green algae. Envir. expl Bot. 27, 481-488. Wenblad A. and Johansson A. (1980) Aluminium i f6rsurade v/istsvenska sj6ar. Vatten 36, 154-157 (English summary).