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Effect of Chelated Trace Metals on Phosphorus Uptake and Storage in Natural Assemblages of Lake Michigan Phytoplankton
J. Great Lakes Res. 16(1):82-89 Internal. Assoc. Great Lakes Res., 1990
EFFECT OF CHELATED TRACE METALS ON PHOSPHORUS UPTAKE AND STORAGE IN NATURAL ASSEMBLAGES OF LAKE MICHIGAN PHYTOPLANKTON}
Claire L. Schelske2 and Linda Sicko-Goad Center for Great Lakes and Aquatic Sciences The University of Michigan 2200 Bonisteel Blvd. Ann Arbor, Michigan 48109 ABSTRA CT. In experiments with natural phytoplankton assemblages from Lake Michigan, additions of chelated trace metals and orthophosphate increased phosphate uptake more than additions of orthophosphate alone. Enhanced phosphate uptake is attributed to the storage ofpolyphosphate by phytoplankton which can be triggered by high concentrations oftrace metals. Similar effects were obtained with a surface phytoplankton assemblage collected in April and with a metalimnetic assemblage collected in September. Microscopic examination showed that polyphosphate formation was enhanced in populations of Melosira islandica and Scenedesmus opoliensis. These results and data from the literature suggest that polyphosphate storage may play an important role in the phytoplankton population dynamics of Lake Michigan, especially in bays and nearshore areas where tributary inputs ofphosphorus and trace metals are high. If observed effects operate selectively at the population level, populations that accumulate and store phosphorus in nearshore areas of high phosphorus supply may have a competitive advantage over other populations in the nearshore or when transported to the offshore waters of this phosphorus-limited system. INDEX WORDS: Eutrophication, aquatic productivity, nutrients, Lake Michigan, phosphorus, trace metals, phytoplankton.
phytoplankton growth in nearshore waters adjacent to large sources of tributary loading. Although often triggered by phosphorus resup~ ply to phosphate-limited cells, polyphosphate formation also may be triggered by other changes in environmental chemistry including excesses or deficiencies in required nutrients or the presence of potentially toxic inorganic chemicals (e.g., Harold 1966, Lawry and Jensen 1979, Sicko-Goad and Lazinsky 1986). Previous studies have demonstrated that polyphosphate bodies are much more abundant in nearshore assemblages of phytoplankton than in offshore assemblages in Saginaw Bay and southern Lake Huron (Stoermer et al. 1980, Sicko-Goad and Lazinsky 1982). Polyphosphate formation in phytoplankton assemblages in nearshore areas of the Great Lakes, therefore, may result from exposure to a combination of high anthropogenic inputs of phosphorus or other chemical or nutrient excesses. In view of chemical variability and complexity
INTRODUCTION Growth of offshore phytoplankton in the upper Laurentian Great Lakes is phosphorus-limited (see Sche1ske et al. 1986). Nearshore phytoplankton in Lake Michigan, however, especially in the southern basin, may grow in an environment where anthropogenic sources of both nutrients and trace metals are variable and concentrations are much higher than in offshore waters. Concentrations of phosphorus (Sche1ske et al. 1980) and trace metals (Robbins et al. 1972) in the Grand River and other tributaries to the southern basin may be an order of magnitude greater than in the open lake. Phosphorus, therefore, would not be expected to limit [Contribution No. 522 of the Center for Great Lakes and Aquatic Sciences 2Present Address: Department of Fisheries and Aquaculture University of Florida 7922 NW 7lst Street Gainesville, FL 32606
82
83
TRACE METAL EFFECTS ON PHOSPHORUS UPTAKE TABLE 1. Comparison of physical and chemical characteristics of water sampled at two stations with annual ranges for nearshore and offshore epilimnetic waters (from Schelske et al. 1980). Depths of collection were 1.0 m in April and 27 m in September.
8 April 12 62
Distance Offshore (km) Station Depth (m) Sampling Depth (m) Temperature Cc) pH Total Alkalinity (meq/L) Soluble Reactive P (fLg/L) Total P (p.g/L)
2 1
0.8 17.3 1.57 0.38 2.84
Si0 2 (mg/L) N0 3-N (mg/L) Chlorophyll a (p.g/L)
Secchi Disc (m) *From Rousar (1973). ND
=
2.5 not detected.
of the nearshore environment, experiments were designed to determine if trace metal additions could stimulate phosphorus uptake in natural assemblages of phytoplankton from Lake Michigan. Two types of assemblages were studied: a surface assemblage collected in April and a metalimnetic assemblage collected in September. MATERIALS AND METHODS Water samples were collected 8 April and 24 September 1979 with Niskin bottles at stations located west of the Grand River, Grand Haven, Michigan. Collections were made at dusk to minimize effects of light shock on sensitive phytoplankton populations. Water was transported in insulated boxes to the laboratory to minimize temperature changes and to avoid exposure to light. Nutrient concentrations at both stations were within the ranges reported previously in the literature (Table 1). Levels of total phosphorus, silica, and nitrate nitrogen at the station located 12 km offshore (April experiment) were in the range for nearshore waters whereas these nutrients at the station located 24 km offshore (September experiment) were in the range for offshore waters. In April the surface water temperature was approximately 1°C. In September the surface water temperature was 17°C and the mixed layer depth was approximately 20 m. Diatoms dominated phytoplankton assemblages at both stations. In the laboratory, water was transferred to 2-
24 September 24 95 27 10 0.6 8.3 0.35 0.20 1.36 4.0
Nearshore Waters
Offshore Waters
0.1-24 7.9-9.1 2.1-2.2 ND-l* 15-150 0.1-3.0 0.1-1.0 2->20
0.1-24 8.1-9.0 2.0-2.2 ND-5* 5-10 0.1-1.4 0.1-0.28 0.7-4.5
liter polyethylene containers, treated with different combinations of phosphorus and trace metals, and incubated in a growth chamber on a 12/12 h light! dark photocycle with cool white illumination of 100 ItEin/m2 /sec. Temperature was set at the ambient water temperature at the time of collection. The initial experimental procedures were completed early in the morning on the day after water was collected. Two treatments of phosphorus and trace metals in addition to ambient levels were utilized in each experiment (Table 2). All treatments were run in duplicate. Soluble reactive phosphorus was measured daily with a Technicon AutoAnalyzer system. Phosphorus was added daily, if neces-
TABLE 2. Concentrations of phosphorus and trace metals used in spike experiments. All concentrations are expressed as fLglL except EDTA which is in mg/L. Data from Rossmann (1984, 1986) are ranges ofaverages from offshore waters and waters in southern Lake Michigan which are representative of nearshore conditions.
Chemical Source KH 2P0 4
CoC12 '6H20 McCl 2 '4H 20 Na2Mo04'2H20 ZnCl2 FeCI 3'6H 20 Na2EDTA'2H 20
Final Concentration Element Low High Rossmann P 1,2 8 0.05- 1.7 Co 1 10 Mn 1 10 0.15- 0.5 Mo 1 10 1.3 -10 Zn 5 50 0.48- 6.7 Fe 10 100 2.5 - 9.5 EDTA 0.2 2.0
84
SCHELSKE and SICKO-GOAD
sary, to adjust the concentration in each treatment to the initial designated level. The low phosphorus addition was set at 1 JLg P /L (0.03 JLM) in April. No measurable orthophosphate was present in treatment flasks at the low phosphorus level (l JLg P /L) the day after additions. Only at the high phosphorus treatment were we able to maintain an excess of orthophosphate in the water. The low level, therefore, was increased to 2 JLg P /L (0.06 JLM) in September because of technical difficulties in measuring 1 JLg P /L. Trace metals and EDTA were added as a spike. In the first experiment each trace metal addition and EDTA were added separately but in the second experiment trace metals and EDTA were combined as a single treatment to ensure that trace metals were not precipitated. The high level of trace metals and EDTA was 10 times greater than the low level (Table 2). Three types of experiments were run to determine the importance of physical-chemical processes in relation to metabolic phosphorus uptake. First, trace metals were added at three different time combinations: only on day 0, only on day 4, and on days 0 and 4. Second, phosphorus, trace metals, and EDTA were added in different sequences to filtered lake water to determine if phosphorus was precipitated. Finally, parallel light and dark treatments were used to determine if metabolic uptake or adsorption were involved. Chlorophyll a was measured daily in all replicates to determine if treatments affected chlorophyll production. Chlorophyll was measured fluorometrically on samples collected on HA Millipore filters and extracted for at least 24 hours in the dark in buffered 90070 acetone. Samples were then acidified and measured again to correct for phaeophytin. Particulate phosphorus concentrations obtained from samples collected on day 5 in the April experiment and day 3 in the September experiment were used to evaluate responses to treatments. Particulate phosphorus was determined by difference from measurements of total phosphorus and total soluble phosphorus. Samples for total soluble phosphorus were filtered with prerinsed and presoaked HA Millipore filters (0.45 JLm pore size). All samples were digested with persulfate (Menzel and Corwin 1965) prior to analysis for soluble reactive phosphorus with a Technicon AutoAnalyzero During digestion, samples were concentrated from 25 mL to 15 mL. Data were normalized by dividing particulate phosphorus concentration by
TABLE 3. Chlorophyll a concentrations (p,g/L) on day 5 for the April experiment. Reported values are ranges of two replicates. Averages are the mean ± 1 standard deviation of all replicates for six treatments. Average concentration on day 0 was 2.84 p.g/L. Timing of Trace Metal Addition None Day 0 Low TM Day 0 High TM Day 4 Low TM Day 4 High TM Day 0 + 4 Low TM Day 0 + 4 High TM Average All Low TM Average All High TM
Phosphorus Addition None
Low
4.38-4.50 4.99-4.99 4.62-4.62 5.35-5.97 4.14-4.62 4.99-5.11 5.85-5.85 4.74-5.23 5.85-4.87 5.23-5.85 5.62±0.43 5.19±0.37
High 5.11-6.36 6.14-6.36 5.83-6.64 6.14-6.38 6.14-6.52 6.62-6.64 5.85-6.62 6.45±0.29 6.27±0.38
chlorophyll a concentration because of the treatment effect on chlorophyll a production in the April experiment. The cytochemical method of Ebel et al. (1958) was used to examine phytoplankton for the presence of polyphosphates. This lead sulfide precipitation technique is pH controlled and selectively stains polyphosphates that average chain length 8 or longer and are regarded as acid insoluble polyphosphates. RESULTS In the April experiment, chlorophyll levels were affected by treatments beginning at least by day 4. Chlorophyll a data for day 5 show three basic trends (Table 3). First, average chlorophyll a values for high phosphorus treatments were significantly greater than for low phosphorus treatments for all combinations of trace metal additions (t-test P < .05). Second, both high and low trace metal additions stimulated growth at either low or high phosphorus levels in comparison to the growth obtained with no added trace metals. (High trace metals in combination with either low or high phosphorus levels, however, did not result in higher chlorophyll levels than the parallel treatment with low trace metals.) Third, the average chlorophyll a concentration in the control treatment (no added phosphorus or trace metals) was at least 50070 larger than the average initial concentration on day O. Because chlorophyll concentrations increased in the April experiment, the September experiment was terminated on day 3. With the exception of treatments with low phosphorus, chlorophyll a
85
TRACE METAL EFFECTS ON PHOSPHORUS UPTAKE
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FIG. 1. Average particulate phosphorus concentrations (p,g PIL) on day 5 in April experiment. Average concentration on day 0 was 13.0 p,g PIL. Error bars represent half the range of duplicate values (the other half is hidden).
FIG. 2. Average normalized particulate phosphorus (p,g Plp,g Chi a) on day 5 in April experiment. Error bars represent half the range of duplicate values (the other half is hidden).
decreased generally during the 3-day experiment from a mean ± one standard deviation of 1.36 ± 0.14 (N = 30) for all treatments. The mean and standard deviation of chlorophyll a in treatments with low phosphorus was 1.27 ± 0.12 (N = 6). Other means and standard deviations were: 0.98 ± 0.25 (N = 6), no added phosphorus, and 0.90 ± 0.26 (N = 6), high phosphorus. In the dark treatments means and standard deviations were: 0.98 ± 0.08 (N = 6), low phosphorus, and 0.90 ± 0.14 (N = 6), high phosphorus. In the April experiment, particulate phosphorus concentrations increased with levels of phosphorus and with levels of trace metals only at high phosphorus (Fig. 1). Normalized particulate phosphorus also increased with increasing phosphorus supply and with trace metals at the high level of phosphorus (Fig. 2). Results of one-way analysis of variance showed that significant differences in normalized particulate phosphorus concentration occurred for all treatments with high phosphorus but not with low phosphorus (P < .05). In the September experiment, particulate phosphorus increased generally with phosphorus level for each trace metal treatment with the largest dif-
ference resulting from either low or high additions of phosphorus (Fig. 3). Two-way analysis of variance on the results showed that the main effects due to phosphorus and trace metal additions were significant (P < .01). The greatest effect resulted from the combination of high trace metals and high phosphorus when particulate phosphorus was normalized to chlorophyll (Fig. 4). The relatively low normalized value for the low trace metal and low phosphorus treatment was associated with the highest average chlorophyll concentration for low phosphorus treatments. Examining the raw data revealed that two of the three highest chlorophyll concentrations on day 3 were in this treatment. In the September experiment, light uptake of phosphorus was greater than dark uptake in each treatment with the exception of one outlier, a replicate of the high phosphorus treatment. The increase in normalized particulate phosphorus corrected for dark uptake was greater for high phosphorus treatments than for low phosphorus treatments for all levels of trace metals, but the greatest increase occurred for the combination of high trace metals and high phosphorus. Timing of trace metal additions, whether added
86
SCHELSKE and SICKO-GOAD
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FIG. 3. Average particulate phosphorus concentrations (p.g PIL) on day 3 in September experiment. Average concentration on day 0 was 5.27 p,g PIL. Error bars represent half the range of duplicate values (the other half is hidden).
FIG. 4. Average normalized particulate phosphorus (p.g Plp,g Chi a) on day 3 in September experiment. Error bars represent half the range of duplicate values (the other half is hidden).
on day 0 or day 4, did not affect final particulate phosphorus concentration (Table 4). In addition, final particulate phosphorus concentrations were similar within treatments whether trace metals were added once or twice. In the April experiment, adding high levels of phosphorus and trace metals to filtered lake water resulted in an increase in particulate phosphorus that averaged 1.8 p,g P /L greater than the control. Phytoplankton assemblages from all experiments were examined cytologically for the presence of polyphosphate bodies. Numerous polyphosphate bodies were present in cells from treatments with added phosphorus and trace metals. Polyphosphate bodies were especially prominent in Melosira islandica and Scenedesmus opoliensis (Fig. 5) whereas they were absent or considerably reduced in number and size in control samples.
can be used to support the hypothesis that an increase in particulate phosphorus resulted from biological uptake and polyphosphate formation in phytoplankton in populations and not from physical-chemical processes. First, cytological evidence showed that polyphosphate bodies became more numerous and
DISCUSSION Our results indicate that phosphate uptake in natural phytoplankton assemblages was enhanced by the addition of trace metals and EDTA in the presence of surplus orthophosphate. Four arguments
TABLE 4. Average normalized particulate phosphorus (p.g PIp,g Chi a) for phosphorus vs. trace metals on day 5 of April experiment. Reported values are the average of two replicates. Trace Metal Addition None Day 0 Day 0 Day 4 Day 4 Day 0 Day 0
Phosphorus Addition None Low High 2.55
Low TM High TM Low TM High TM + 4 Low TM + 4 High TM
2.46 2.77
3.09 2.67 3.06 2.60 3.29 2.65 2.62
3.36 3.90 4.52 3.97 4.22 3.30 4.54
TRACE METAL EFFECTS ON PHOSPHORUS UPTAKE
FIG. 5. Light micrographs ofsamples from April experiment. Samples were stained for polyphosphates by the lead-sulfide method (Ebel et aI. 1958). Upper leftMelosira islandicafrom Grand River control. Note several small polyphosphate bodies near central vacuole (arrows). Upper middle- Stephanodiscus sp., also from Grand River with no apparent polyphosphate accumulation. Upper right - Melosira sp. - polyphosphate stain control. Cells incubated in lead nitrate but not treated with ammonium sulfide. No apparent polyphosphate staining. Lower row (left to right) - Melosira sp., Scenedesmus opoliensis, and Melosira islandicafrom the high trace metals with high phosphate treatment. Cells have numerous polyphosphate bodies throughout cytoplasm (arrows), especially near central vacuole in diatoms.
larger in some populations, particularly Melosira islandica and Scenedesmus opoliensis, after treatment with trace metals and phosphorus. The cytochemical method (Ebel et al. 1958) has been used routinely with light microscopy to confirm the
87
presence of polyphosphate inclusions (Jensen 1968, 1969). Second, additions of high levels of trace metals and phosphorus to filtered lake water resulted in an increase in particulate phosphorus that averaged 1.8 p,g P/L, a small increase compared with the 16 p,g P/L average increase obtained for the high phosphorus and high trace metal treatment of lake water with its natural phytoplankton assemblage. Third, similar increases in particulate phosphorus occurred whether trace metals were added on day 0, on day 4, or on both day 0 and day 4. If physical-chemical effects were important in precipitating phosphorus, adding trace metals twice (days 0 and 4) should have increased the concentration of particulate phosphorus, especially in the high phosphorus treatment. Finally, dark uptake of phosphorus with the exception of one replicate was less than light uptake, suggesting a metabolically mediated process and not a physical-chemical process. Chelated trace metals stimulated phosphate uptake in phytoplankton assemblages that were collected in two seasons under different environmental conditions. In April the assemblage was collected from a homothermous water column where growth was primarily light limited because the mixing depth of the water column was at least several fold greater than the photic depth. In addition, under controlled light in the laboratory, assemblage growth, as measured by chlorophyll a, occurred in the control treatment. In September the assemblage was collected at 27 m where nutrient-light interactions would have been important in phytoplankton dynamics (Fahnenstiel et al. 1984, Schelske et al. 1984). During the experiment, chlorophyll concentrations decreased except in low phosphorus additions. Such decreases in chlorophyll are not unexpected because they have been observed previously in the initial 2 or 3 days of other experiments with natural phytoplankton assemblages (see Schelske 1984). Mechanisms for these decreases have not been identified, but phytoplankton populations eventually respond with logarithmic growth if nutrient or other limiting factors are removed. EDTA added with trace metals was present in excess of amounts required to complex the trace metals added initially and especially with the high treatment of EDTA to complex levels of soluble metals that have been reported previously (Table 2). Determining how much metal is complexed,
88
SCHELSKE and SICKO-GOAD
however, would be complicated by the presence of high levels of trace metals in particulate form (Rossmann 1984, 1986). Some of these particulate trace metals may be sequestered by added chelating agents. Waters of Lake Michigan are buffered mainly with calcium carbonate. Average concentrations of major cations and anions in addition to those in Table 2 are 35 mg CalL, 11 mg MgIL, 4.8 mg NaiL, 1.1 mg KIL, 22 mg S04/L, and 8 mg Cl/L (Bartone and Schelske 1982). Given the excess of EDTA added, chemical precipitation of phosphorus with trace metals would not be expected. EDTA alone may stimulate algal growth by increasing availability of nutrients or by decreasing toxicity of trace metals and other inhibitory substances (Smayda 1974, Maestrini et al. 1984). In experiments with natural phytoplankton assemblages from the Great Lakes, EDTA alone stimulated phytoplankton growth and chlorophyll production (see Lin and Schelske 1981, Schelske 1984). EDTA was used in our experiments to maintain trace metals in solution. Whether polyphosphate formation was triggered by chelated trace metals, by EDTA alone, or by other forms of trace metals was not tested. In both experiments, the effect of chelated trace metals on phosphorus uptake by phytoplankton was obvious in the treatment with high trace metals and high phosphorus. These high levels, however, could occur in the nearshore waters of Lake Michigan. High levels of trace metals utilized in our experiments are within the range and for some metals are lower than values reported for tributary inputs (Robbins et al. 1972). The high level of phosphorus is equal to the average total phosphorus concentration of 7.5 to 8.0 I1g P IL (0.24 to 0.26 11M) in offshore waters (Bartone and Sche1ske 1982, Schelske et al. 1980). We found a concentration of total phosphorus at the station located at 12 km offshore that was about twice greater than the maximum expected for offshore waters (Table 1). Tarapchak and Rubitschun (1981) sampled seasonally and found levels of orthophosphorus at a station located 15 km from the Grand River that were higher than expected for offshore waters. These elevated levels of phosphorus can be attributed to offshore transport of nutrients from nearshore sources. We believe that phytoplankton populations in large areas of the nearshore zone of southeastern Lake Michigan may be affected directly or indirectly by high loads of phosphorus from the Grand
River and other large tributaries. Logarithmic growth of some river populations may be sustained at low phosphorus supplies by utilization of stored polyphosphates under experimental conditions that simulate offshore transport (Schelske et al. 1984). It has been demonstrated previously that polyphosphate storage is more extensive in nearshore algal populations than in offshore populations (Stoermer et al. 1980, Sicko-Goad and Lazinsky 1982). Our results demonstrate that relatively small additions of phosphorus and chelated trace metals may stimulate polyphosphate formation selectively in natural phytoplankton assemblages. Trace metals from tributaries with high anthropogenic loadings, therefore, may play an important role in phosphorus uptake and partitioning among phytoplankton populations and thus affect resource competition and succession among phytoplankton populations in waters that are influenced by high phosphorus loadings. REFERENCES Bartone, C. R., and Schelske, C. L. 1982. Lake-wide seasonal changes in limnological conditions in Lake Michigan in 1976. J. Great Lakes Res. 8:413-427. Ebel, J. P., Colas, J., and Muller, S. 1958. Recherches cytochimiques sur les polyphosphates. II. Mise au point de methodes de detection cytochimiques specifiques des polyphosphates. Exptl. Cell Res. 15: 28-36. Fahnenstiel, G. L., Scavia, D., and Schelske, C. L. 1984. Nutrient-light interactions in the Lake Michigan subsurface chlorophyll layer. Verh. Internat. Verein. Limnol. 22:440-444. Harold, F. M. 1966. Inorganic polyphosphates in biology: structure, metabolism, and function. Bacteriol. Rev. 30:772-794. Jensen, T. E. 1968. Electron microscopy of polyphosphate bodies in a blue-green alga, Nostoc pruniforme. Archiv. Mikrobiol. 62:144-152. _ _ _ _ . 1969. Fine structure of developing polyphosphate bodies in a blue-green alga, Plectonema boryanum. Archiv. Mikrobiol. 67:328-338. Lawry, N. H., and Jensen, T. E. 1979. Deposition of condensed phosphate as an effect of varying sulfur deficiency in the Cyanobacterium Synechococcus sp (Anacystis nidulans). Arch. Microbiol. 120:1-7. Lin, C. K., and Schelske, C. L. 1981. Seasonal variation of potential nutrient limitation to chlorophyll production in Southern Lake Huron. Can. J. Fish. Aquat. Sci. 38:1-9. Maestrini, S. Y., Bonin, D. J., and Droop, M. R. 1984. Phytoplankton as indicators of sea water quality: bioassay approaches and protocols. In L. E. Shubert
TRACE METAL EFFECTS ON PHOSPHORUS UPTAKE (ed.), Algae as Ecological Indicators, pp. 71-132. Academic Press, Inc. (London) Limited. Menzel, D. W., and Corwin, N. 1965. The measurement of total phosphorus in seawater based on the liberation of organically bound fractions by persulfate oxidation. Limnol. Oceanogr. 10:280-282. Robbins, J. A., Landstrom, E., and Wahlgren, M. 1972. Tributary inputs of soluble trace metals to Lake Michigan. In Proc. 15th ConI. Great Lakes Res., pp. 270-290. Internat. Assoc. Great Lakes Res. Rousar, D. C. 1973. Seasonal and spatial changes in primary production and nutrients in Lake Michigan. Water Air Soil Pollut. 2:497-514. Rossmann, R. 1984. Trace metal concentrations in the offshore waters of Lakes Erie and Michigan. Spec. Rept. No. 108, Great Lakes Res. Div., University of Michigan. _ _ _ _ . 1986. Waters of southeastern nearshore Lake Michigan, pp. 51-85. In R. Rossmann (ed), Impact of the Donald C. Cook Nuclear Plant. University of Michigan, Great Lakes Res. Div. Pub. 22. Schelske, C. L. 1984. In situ and natural phytoplankton assemblage bioassays. In L. E. Shubert (ed), Algae as Ecological Indicators, pp. 15-47. Academic Press, Inc. (London) Limited. _ _ _ _ , Feldt, L. E., and Simmons, M. S. 1980.
Phytoplankton and physical-chemical conditions in selected rivers and the coastal zone of Lake Michigan, 1972. University of Michigan, Great Lakes Res. Div. Pub. 19. _ _ _ _ , Davis, C.O., and Feldt, L. E., 1984. Growth responses of river and lake phytoplankton
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populations in Lake Michigan water. Verh.Internat. Verein. Limnol. 22:445-451. _ _ _ _ , Stoermer, E. E, Fahnenstiel, G. L., and Haibach, M. 1986. Phosphorus enrichment, silica utilization, and biogeochemical silica depletion in the Great Lakes. Can. J. Fish. Aquat. Sci. 43:407-415. Sicko-Goad, L., and Lazinsky, D. 1982. Polyphosphate body formation and degradation in Plectonema boryanum (Cyanophyceae). Micron 13:459-460. _ _ _ _ , and Lazinsky, D. 1986. Quantitative ultrastructural changes associated with lead-coupled luxury phosphate uptake and polyphosphate utilization. Arch. Environ. Contam. Toxicol. 15:617-627. Smayda, T. J. 1974. Bioassay of the growth potential of the surface water of lower Narragansett Bay over an annual cycle using the diatom Thalassiosira pseudonana (oceanic clone, 13-1). Limnol. Oceanogr. 19:889-901. Stoermer, E. E, Sicko-Goad, L., and Lazinsky, D., 1980. Synergistic effects of phosphorus and trace metal loadings on Great Lakes phytoplankton. In
Proceedings of the Third USA-USSR Symposium on the effects ofpollutants upon aquatic systems: theoretical aspects of aquatic toxicology, pp. 171-186. U.S. Environmental Protection Agency, Duluth, Minnesota. Tarapchak, S. J., and Rubitschun, C. 1981. Comparisons of soluble reactive phosphorus and orthophosphorus concentrations at an offshore station in southern Lake Michigan. J. Great Lakes Res. 7: 290-298.
EFFECT OF CHELATED TRACE METALS ON PHOSPHORUS UPTAKE AND STORAGE IN NATURAL ASSEMBLAGES OF LAKE MICHIGAN PHYTOPLANKTON}
Claire L. Schelske2 and Linda Sicko-Goad Center for Great Lakes and Aquatic Sciences The University of Michigan 2200 Bonisteel Blvd. Ann Arbor, Michigan 48109 ABSTRA CT. In experiments with natural phytoplankton assemblages from Lake Michigan, additions of chelated trace metals and orthophosphate increased phosphate uptake more than additions of orthophosphate alone. Enhanced phosphate uptake is attributed to the storage ofpolyphosphate by phytoplankton which can be triggered by high concentrations oftrace metals. Similar effects were obtained with a surface phytoplankton assemblage collected in April and with a metalimnetic assemblage collected in September. Microscopic examination showed that polyphosphate formation was enhanced in populations of Melosira islandica and Scenedesmus opoliensis. These results and data from the literature suggest that polyphosphate storage may play an important role in the phytoplankton population dynamics of Lake Michigan, especially in bays and nearshore areas where tributary inputs ofphosphorus and trace metals are high. If observed effects operate selectively at the population level, populations that accumulate and store phosphorus in nearshore areas of high phosphorus supply may have a competitive advantage over other populations in the nearshore or when transported to the offshore waters of this phosphorus-limited system. INDEX WORDS: Eutrophication, aquatic productivity, nutrients, Lake Michigan, phosphorus, trace metals, phytoplankton.
phytoplankton growth in nearshore waters adjacent to large sources of tributary loading. Although often triggered by phosphorus resup~ ply to phosphate-limited cells, polyphosphate formation also may be triggered by other changes in environmental chemistry including excesses or deficiencies in required nutrients or the presence of potentially toxic inorganic chemicals (e.g., Harold 1966, Lawry and Jensen 1979, Sicko-Goad and Lazinsky 1986). Previous studies have demonstrated that polyphosphate bodies are much more abundant in nearshore assemblages of phytoplankton than in offshore assemblages in Saginaw Bay and southern Lake Huron (Stoermer et al. 1980, Sicko-Goad and Lazinsky 1982). Polyphosphate formation in phytoplankton assemblages in nearshore areas of the Great Lakes, therefore, may result from exposure to a combination of high anthropogenic inputs of phosphorus or other chemical or nutrient excesses. In view of chemical variability and complexity
INTRODUCTION Growth of offshore phytoplankton in the upper Laurentian Great Lakes is phosphorus-limited (see Sche1ske et al. 1986). Nearshore phytoplankton in Lake Michigan, however, especially in the southern basin, may grow in an environment where anthropogenic sources of both nutrients and trace metals are variable and concentrations are much higher than in offshore waters. Concentrations of phosphorus (Sche1ske et al. 1980) and trace metals (Robbins et al. 1972) in the Grand River and other tributaries to the southern basin may be an order of magnitude greater than in the open lake. Phosphorus, therefore, would not be expected to limit [Contribution No. 522 of the Center for Great Lakes and Aquatic Sciences 2Present Address: Department of Fisheries and Aquaculture University of Florida 7922 NW 7lst Street Gainesville, FL 32606
82
83
TRACE METAL EFFECTS ON PHOSPHORUS UPTAKE TABLE 1. Comparison of physical and chemical characteristics of water sampled at two stations with annual ranges for nearshore and offshore epilimnetic waters (from Schelske et al. 1980). Depths of collection were 1.0 m in April and 27 m in September.
8 April 12 62
Distance Offshore (km) Station Depth (m) Sampling Depth (m) Temperature Cc) pH Total Alkalinity (meq/L) Soluble Reactive P (fLg/L) Total P (p.g/L)
2 1
0.8 17.3 1.57 0.38 2.84
Si0 2 (mg/L) N0 3-N (mg/L) Chlorophyll a (p.g/L)
Secchi Disc (m) *From Rousar (1973). ND
=
2.5 not detected.
of the nearshore environment, experiments were designed to determine if trace metal additions could stimulate phosphorus uptake in natural assemblages of phytoplankton from Lake Michigan. Two types of assemblages were studied: a surface assemblage collected in April and a metalimnetic assemblage collected in September. MATERIALS AND METHODS Water samples were collected 8 April and 24 September 1979 with Niskin bottles at stations located west of the Grand River, Grand Haven, Michigan. Collections were made at dusk to minimize effects of light shock on sensitive phytoplankton populations. Water was transported in insulated boxes to the laboratory to minimize temperature changes and to avoid exposure to light. Nutrient concentrations at both stations were within the ranges reported previously in the literature (Table 1). Levels of total phosphorus, silica, and nitrate nitrogen at the station located 12 km offshore (April experiment) were in the range for nearshore waters whereas these nutrients at the station located 24 km offshore (September experiment) were in the range for offshore waters. In April the surface water temperature was approximately 1°C. In September the surface water temperature was 17°C and the mixed layer depth was approximately 20 m. Diatoms dominated phytoplankton assemblages at both stations. In the laboratory, water was transferred to 2-
24 September 24 95 27 10 0.6 8.3 0.35 0.20 1.36 4.0
Nearshore Waters
Offshore Waters
0.1-24 7.9-9.1 2.1-2.2 ND-l* 15-150 0.1-3.0 0.1-1.0 2->20
0.1-24 8.1-9.0 2.0-2.2 ND-5* 5-10 0.1-1.4 0.1-0.28 0.7-4.5
liter polyethylene containers, treated with different combinations of phosphorus and trace metals, and incubated in a growth chamber on a 12/12 h light! dark photocycle with cool white illumination of 100 ItEin/m2 /sec. Temperature was set at the ambient water temperature at the time of collection. The initial experimental procedures were completed early in the morning on the day after water was collected. Two treatments of phosphorus and trace metals in addition to ambient levels were utilized in each experiment (Table 2). All treatments were run in duplicate. Soluble reactive phosphorus was measured daily with a Technicon AutoAnalyzer system. Phosphorus was added daily, if neces-
TABLE 2. Concentrations of phosphorus and trace metals used in spike experiments. All concentrations are expressed as fLglL except EDTA which is in mg/L. Data from Rossmann (1984, 1986) are ranges ofaverages from offshore waters and waters in southern Lake Michigan which are representative of nearshore conditions.
Chemical Source KH 2P0 4
CoC12 '6H20 McCl 2 '4H 20 Na2Mo04'2H20 ZnCl2 FeCI 3'6H 20 Na2EDTA'2H 20
Final Concentration Element Low High Rossmann P 1,2 8 0.05- 1.7 Co 1 10 Mn 1 10 0.15- 0.5 Mo 1 10 1.3 -10 Zn 5 50 0.48- 6.7 Fe 10 100 2.5 - 9.5 EDTA 0.2 2.0
84
SCHELSKE and SICKO-GOAD
sary, to adjust the concentration in each treatment to the initial designated level. The low phosphorus addition was set at 1 JLg P /L (0.03 JLM) in April. No measurable orthophosphate was present in treatment flasks at the low phosphorus level (l JLg P /L) the day after additions. Only at the high phosphorus treatment were we able to maintain an excess of orthophosphate in the water. The low level, therefore, was increased to 2 JLg P /L (0.06 JLM) in September because of technical difficulties in measuring 1 JLg P /L. Trace metals and EDTA were added as a spike. In the first experiment each trace metal addition and EDTA were added separately but in the second experiment trace metals and EDTA were combined as a single treatment to ensure that trace metals were not precipitated. The high level of trace metals and EDTA was 10 times greater than the low level (Table 2). Three types of experiments were run to determine the importance of physical-chemical processes in relation to metabolic phosphorus uptake. First, trace metals were added at three different time combinations: only on day 0, only on day 4, and on days 0 and 4. Second, phosphorus, trace metals, and EDTA were added in different sequences to filtered lake water to determine if phosphorus was precipitated. Finally, parallel light and dark treatments were used to determine if metabolic uptake or adsorption were involved. Chlorophyll a was measured daily in all replicates to determine if treatments affected chlorophyll production. Chlorophyll was measured fluorometrically on samples collected on HA Millipore filters and extracted for at least 24 hours in the dark in buffered 90070 acetone. Samples were then acidified and measured again to correct for phaeophytin. Particulate phosphorus concentrations obtained from samples collected on day 5 in the April experiment and day 3 in the September experiment were used to evaluate responses to treatments. Particulate phosphorus was determined by difference from measurements of total phosphorus and total soluble phosphorus. Samples for total soluble phosphorus were filtered with prerinsed and presoaked HA Millipore filters (0.45 JLm pore size). All samples were digested with persulfate (Menzel and Corwin 1965) prior to analysis for soluble reactive phosphorus with a Technicon AutoAnalyzero During digestion, samples were concentrated from 25 mL to 15 mL. Data were normalized by dividing particulate phosphorus concentration by
TABLE 3. Chlorophyll a concentrations (p,g/L) on day 5 for the April experiment. Reported values are ranges of two replicates. Averages are the mean ± 1 standard deviation of all replicates for six treatments. Average concentration on day 0 was 2.84 p.g/L. Timing of Trace Metal Addition None Day 0 Low TM Day 0 High TM Day 4 Low TM Day 4 High TM Day 0 + 4 Low TM Day 0 + 4 High TM Average All Low TM Average All High TM
Phosphorus Addition None
Low
4.38-4.50 4.99-4.99 4.62-4.62 5.35-5.97 4.14-4.62 4.99-5.11 5.85-5.85 4.74-5.23 5.85-4.87 5.23-5.85 5.62±0.43 5.19±0.37
High 5.11-6.36 6.14-6.36 5.83-6.64 6.14-6.38 6.14-6.52 6.62-6.64 5.85-6.62 6.45±0.29 6.27±0.38
chlorophyll a concentration because of the treatment effect on chlorophyll a production in the April experiment. The cytochemical method of Ebel et al. (1958) was used to examine phytoplankton for the presence of polyphosphates. This lead sulfide precipitation technique is pH controlled and selectively stains polyphosphates that average chain length 8 or longer and are regarded as acid insoluble polyphosphates. RESULTS In the April experiment, chlorophyll levels were affected by treatments beginning at least by day 4. Chlorophyll a data for day 5 show three basic trends (Table 3). First, average chlorophyll a values for high phosphorus treatments were significantly greater than for low phosphorus treatments for all combinations of trace metal additions (t-test P < .05). Second, both high and low trace metal additions stimulated growth at either low or high phosphorus levels in comparison to the growth obtained with no added trace metals. (High trace metals in combination with either low or high phosphorus levels, however, did not result in higher chlorophyll levels than the parallel treatment with low trace metals.) Third, the average chlorophyll a concentration in the control treatment (no added phosphorus or trace metals) was at least 50070 larger than the average initial concentration on day O. Because chlorophyll concentrations increased in the April experiment, the September experiment was terminated on day 3. With the exception of treatments with low phosphorus, chlorophyll a
85
TRACE METAL EFFECTS ON PHOSPHORUS UPTAKE
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FIG. 1. Average particulate phosphorus concentrations (p,g PIL) on day 5 in April experiment. Average concentration on day 0 was 13.0 p,g PIL. Error bars represent half the range of duplicate values (the other half is hidden).
FIG. 2. Average normalized particulate phosphorus (p,g Plp,g Chi a) on day 5 in April experiment. Error bars represent half the range of duplicate values (the other half is hidden).
decreased generally during the 3-day experiment from a mean ± one standard deviation of 1.36 ± 0.14 (N = 30) for all treatments. The mean and standard deviation of chlorophyll a in treatments with low phosphorus was 1.27 ± 0.12 (N = 6). Other means and standard deviations were: 0.98 ± 0.25 (N = 6), no added phosphorus, and 0.90 ± 0.26 (N = 6), high phosphorus. In the dark treatments means and standard deviations were: 0.98 ± 0.08 (N = 6), low phosphorus, and 0.90 ± 0.14 (N = 6), high phosphorus. In the April experiment, particulate phosphorus concentrations increased with levels of phosphorus and with levels of trace metals only at high phosphorus (Fig. 1). Normalized particulate phosphorus also increased with increasing phosphorus supply and with trace metals at the high level of phosphorus (Fig. 2). Results of one-way analysis of variance showed that significant differences in normalized particulate phosphorus concentration occurred for all treatments with high phosphorus but not with low phosphorus (P < .05). In the September experiment, particulate phosphorus increased generally with phosphorus level for each trace metal treatment with the largest dif-
ference resulting from either low or high additions of phosphorus (Fig. 3). Two-way analysis of variance on the results showed that the main effects due to phosphorus and trace metal additions were significant (P < .01). The greatest effect resulted from the combination of high trace metals and high phosphorus when particulate phosphorus was normalized to chlorophyll (Fig. 4). The relatively low normalized value for the low trace metal and low phosphorus treatment was associated with the highest average chlorophyll concentration for low phosphorus treatments. Examining the raw data revealed that two of the three highest chlorophyll concentrations on day 3 were in this treatment. In the September experiment, light uptake of phosphorus was greater than dark uptake in each treatment with the exception of one outlier, a replicate of the high phosphorus treatment. The increase in normalized particulate phosphorus corrected for dark uptake was greater for high phosphorus treatments than for low phosphorus treatments for all levels of trace metals, but the greatest increase occurred for the combination of high trace metals and high phosphorus. Timing of trace metal additions, whether added
86
SCHELSKE and SICKO-GOAD
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FIG. 3. Average particulate phosphorus concentrations (p.g PIL) on day 3 in September experiment. Average concentration on day 0 was 5.27 p,g PIL. Error bars represent half the range of duplicate values (the other half is hidden).
FIG. 4. Average normalized particulate phosphorus (p.g Plp,g Chi a) on day 3 in September experiment. Error bars represent half the range of duplicate values (the other half is hidden).
on day 0 or day 4, did not affect final particulate phosphorus concentration (Table 4). In addition, final particulate phosphorus concentrations were similar within treatments whether trace metals were added once or twice. In the April experiment, adding high levels of phosphorus and trace metals to filtered lake water resulted in an increase in particulate phosphorus that averaged 1.8 p,g P /L greater than the control. Phytoplankton assemblages from all experiments were examined cytologically for the presence of polyphosphate bodies. Numerous polyphosphate bodies were present in cells from treatments with added phosphorus and trace metals. Polyphosphate bodies were especially prominent in Melosira islandica and Scenedesmus opoliensis (Fig. 5) whereas they were absent or considerably reduced in number and size in control samples.
can be used to support the hypothesis that an increase in particulate phosphorus resulted from biological uptake and polyphosphate formation in phytoplankton in populations and not from physical-chemical processes. First, cytological evidence showed that polyphosphate bodies became more numerous and
DISCUSSION Our results indicate that phosphate uptake in natural phytoplankton assemblages was enhanced by the addition of trace metals and EDTA in the presence of surplus orthophosphate. Four arguments
TABLE 4. Average normalized particulate phosphorus (p.g PIp,g Chi a) for phosphorus vs. trace metals on day 5 of April experiment. Reported values are the average of two replicates. Trace Metal Addition None Day 0 Day 0 Day 4 Day 4 Day 0 Day 0
Phosphorus Addition None Low High 2.55
Low TM High TM Low TM High TM + 4 Low TM + 4 High TM
2.46 2.77
3.09 2.67 3.06 2.60 3.29 2.65 2.62
3.36 3.90 4.52 3.97 4.22 3.30 4.54
TRACE METAL EFFECTS ON PHOSPHORUS UPTAKE
FIG. 5. Light micrographs ofsamples from April experiment. Samples were stained for polyphosphates by the lead-sulfide method (Ebel et aI. 1958). Upper leftMelosira islandicafrom Grand River control. Note several small polyphosphate bodies near central vacuole (arrows). Upper middle- Stephanodiscus sp., also from Grand River with no apparent polyphosphate accumulation. Upper right - Melosira sp. - polyphosphate stain control. Cells incubated in lead nitrate but not treated with ammonium sulfide. No apparent polyphosphate staining. Lower row (left to right) - Melosira sp., Scenedesmus opoliensis, and Melosira islandicafrom the high trace metals with high phosphate treatment. Cells have numerous polyphosphate bodies throughout cytoplasm (arrows), especially near central vacuole in diatoms.
larger in some populations, particularly Melosira islandica and Scenedesmus opoliensis, after treatment with trace metals and phosphorus. The cytochemical method (Ebel et al. 1958) has been used routinely with light microscopy to confirm the
87
presence of polyphosphate inclusions (Jensen 1968, 1969). Second, additions of high levels of trace metals and phosphorus to filtered lake water resulted in an increase in particulate phosphorus that averaged 1.8 p,g P/L, a small increase compared with the 16 p,g P/L average increase obtained for the high phosphorus and high trace metal treatment of lake water with its natural phytoplankton assemblage. Third, similar increases in particulate phosphorus occurred whether trace metals were added on day 0, on day 4, or on both day 0 and day 4. If physical-chemical effects were important in precipitating phosphorus, adding trace metals twice (days 0 and 4) should have increased the concentration of particulate phosphorus, especially in the high phosphorus treatment. Finally, dark uptake of phosphorus with the exception of one replicate was less than light uptake, suggesting a metabolically mediated process and not a physical-chemical process. Chelated trace metals stimulated phosphate uptake in phytoplankton assemblages that were collected in two seasons under different environmental conditions. In April the assemblage was collected from a homothermous water column where growth was primarily light limited because the mixing depth of the water column was at least several fold greater than the photic depth. In addition, under controlled light in the laboratory, assemblage growth, as measured by chlorophyll a, occurred in the control treatment. In September the assemblage was collected at 27 m where nutrient-light interactions would have been important in phytoplankton dynamics (Fahnenstiel et al. 1984, Schelske et al. 1984). During the experiment, chlorophyll concentrations decreased except in low phosphorus additions. Such decreases in chlorophyll are not unexpected because they have been observed previously in the initial 2 or 3 days of other experiments with natural phytoplankton assemblages (see Schelske 1984). Mechanisms for these decreases have not been identified, but phytoplankton populations eventually respond with logarithmic growth if nutrient or other limiting factors are removed. EDTA added with trace metals was present in excess of amounts required to complex the trace metals added initially and especially with the high treatment of EDTA to complex levels of soluble metals that have been reported previously (Table 2). Determining how much metal is complexed,
88
SCHELSKE and SICKO-GOAD
however, would be complicated by the presence of high levels of trace metals in particulate form (Rossmann 1984, 1986). Some of these particulate trace metals may be sequestered by added chelating agents. Waters of Lake Michigan are buffered mainly with calcium carbonate. Average concentrations of major cations and anions in addition to those in Table 2 are 35 mg CalL, 11 mg MgIL, 4.8 mg NaiL, 1.1 mg KIL, 22 mg S04/L, and 8 mg Cl/L (Bartone and Schelske 1982). Given the excess of EDTA added, chemical precipitation of phosphorus with trace metals would not be expected. EDTA alone may stimulate algal growth by increasing availability of nutrients or by decreasing toxicity of trace metals and other inhibitory substances (Smayda 1974, Maestrini et al. 1984). In experiments with natural phytoplankton assemblages from the Great Lakes, EDTA alone stimulated phytoplankton growth and chlorophyll production (see Lin and Schelske 1981, Schelske 1984). EDTA was used in our experiments to maintain trace metals in solution. Whether polyphosphate formation was triggered by chelated trace metals, by EDTA alone, or by other forms of trace metals was not tested. In both experiments, the effect of chelated trace metals on phosphorus uptake by phytoplankton was obvious in the treatment with high trace metals and high phosphorus. These high levels, however, could occur in the nearshore waters of Lake Michigan. High levels of trace metals utilized in our experiments are within the range and for some metals are lower than values reported for tributary inputs (Robbins et al. 1972). The high level of phosphorus is equal to the average total phosphorus concentration of 7.5 to 8.0 I1g P IL (0.24 to 0.26 11M) in offshore waters (Bartone and Sche1ske 1982, Schelske et al. 1980). We found a concentration of total phosphorus at the station located at 12 km offshore that was about twice greater than the maximum expected for offshore waters (Table 1). Tarapchak and Rubitschun (1981) sampled seasonally and found levels of orthophosphorus at a station located 15 km from the Grand River that were higher than expected for offshore waters. These elevated levels of phosphorus can be attributed to offshore transport of nutrients from nearshore sources. We believe that phytoplankton populations in large areas of the nearshore zone of southeastern Lake Michigan may be affected directly or indirectly by high loads of phosphorus from the Grand
River and other large tributaries. Logarithmic growth of some river populations may be sustained at low phosphorus supplies by utilization of stored polyphosphates under experimental conditions that simulate offshore transport (Schelske et al. 1984). It has been demonstrated previously that polyphosphate storage is more extensive in nearshore algal populations than in offshore populations (Stoermer et al. 1980, Sicko-Goad and Lazinsky 1982). Our results demonstrate that relatively small additions of phosphorus and chelated trace metals may stimulate polyphosphate formation selectively in natural phytoplankton assemblages. Trace metals from tributaries with high anthropogenic loadings, therefore, may play an important role in phosphorus uptake and partitioning among phytoplankton populations and thus affect resource competition and succession among phytoplankton populations in waters that are influenced by high phosphorus loadings. REFERENCES Bartone, C. R., and Schelske, C. L. 1982. Lake-wide seasonal changes in limnological conditions in Lake Michigan in 1976. J. Great Lakes Res. 8:413-427. Ebel, J. P., Colas, J., and Muller, S. 1958. Recherches cytochimiques sur les polyphosphates. II. Mise au point de methodes de detection cytochimiques specifiques des polyphosphates. Exptl. Cell Res. 15: 28-36. Fahnenstiel, G. L., Scavia, D., and Schelske, C. L. 1984. Nutrient-light interactions in the Lake Michigan subsurface chlorophyll layer. Verh. Internat. Verein. Limnol. 22:440-444. Harold, F. M. 1966. Inorganic polyphosphates in biology: structure, metabolism, and function. Bacteriol. Rev. 30:772-794. Jensen, T. E. 1968. Electron microscopy of polyphosphate bodies in a blue-green alga, Nostoc pruniforme. Archiv. Mikrobiol. 62:144-152. _ _ _ _ . 1969. Fine structure of developing polyphosphate bodies in a blue-green alga, Plectonema boryanum. Archiv. Mikrobiol. 67:328-338. Lawry, N. H., and Jensen, T. E. 1979. Deposition of condensed phosphate as an effect of varying sulfur deficiency in the Cyanobacterium Synechococcus sp (Anacystis nidulans). Arch. Microbiol. 120:1-7. Lin, C. K., and Schelske, C. L. 1981. Seasonal variation of potential nutrient limitation to chlorophyll production in Southern Lake Huron. Can. J. Fish. Aquat. Sci. 38:1-9. Maestrini, S. Y., Bonin, D. J., and Droop, M. R. 1984. Phytoplankton as indicators of sea water quality: bioassay approaches and protocols. In L. E. Shubert
TRACE METAL EFFECTS ON PHOSPHORUS UPTAKE (ed.), Algae as Ecological Indicators, pp. 71-132. Academic Press, Inc. (London) Limited. Menzel, D. W., and Corwin, N. 1965. The measurement of total phosphorus in seawater based on the liberation of organically bound fractions by persulfate oxidation. Limnol. Oceanogr. 10:280-282. Robbins, J. A., Landstrom, E., and Wahlgren, M. 1972. Tributary inputs of soluble trace metals to Lake Michigan. In Proc. 15th ConI. Great Lakes Res., pp. 270-290. Internat. Assoc. Great Lakes Res. Rousar, D. C. 1973. Seasonal and spatial changes in primary production and nutrients in Lake Michigan. Water Air Soil Pollut. 2:497-514. Rossmann, R. 1984. Trace metal concentrations in the offshore waters of Lakes Erie and Michigan. Spec. Rept. No. 108, Great Lakes Res. Div., University of Michigan. _ _ _ _ . 1986. Waters of southeastern nearshore Lake Michigan, pp. 51-85. In R. Rossmann (ed), Impact of the Donald C. Cook Nuclear Plant. University of Michigan, Great Lakes Res. Div. Pub. 22. Schelske, C. L. 1984. In situ and natural phytoplankton assemblage bioassays. In L. E. Shubert (ed), Algae as Ecological Indicators, pp. 15-47. Academic Press, Inc. (London) Limited. _ _ _ _ , Feldt, L. E., and Simmons, M. S. 1980.
Phytoplankton and physical-chemical conditions in selected rivers and the coastal zone of Lake Michigan, 1972. University of Michigan, Great Lakes Res. Div. Pub. 19. _ _ _ _ , Davis, C.O., and Feldt, L. E., 1984. Growth responses of river and lake phytoplankton
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populations in Lake Michigan water. Verh.Internat. Verein. Limnol. 22:445-451. _ _ _ _ , Stoermer, E. E, Fahnenstiel, G. L., and Haibach, M. 1986. Phosphorus enrichment, silica utilization, and biogeochemical silica depletion in the Great Lakes. Can. J. Fish. Aquat. Sci. 43:407-415. Sicko-Goad, L., and Lazinsky, D. 1982. Polyphosphate body formation and degradation in Plectonema boryanum (Cyanophyceae). Micron 13:459-460. _ _ _ _ , and Lazinsky, D. 1986. Quantitative ultrastructural changes associated with lead-coupled luxury phosphate uptake and polyphosphate utilization. Arch. Environ. Contam. Toxicol. 15:617-627. Smayda, T. J. 1974. Bioassay of the growth potential of the surface water of lower Narragansett Bay over an annual cycle using the diatom Thalassiosira pseudonana (oceanic clone, 13-1). Limnol. Oceanogr. 19:889-901. Stoermer, E. E, Sicko-Goad, L., and Lazinsky, D., 1980. Synergistic effects of phosphorus and trace metal loadings on Great Lakes phytoplankton. In
Proceedings of the Third USA-USSR Symposium on the effects ofpollutants upon aquatic systems: theoretical aspects of aquatic toxicology, pp. 171-186. U.S. Environmental Protection Agency, Duluth, Minnesota. Tarapchak, S. J., and Rubitschun, C. 1981. Comparisons of soluble reactive phosphorus and orthophosphorus concentrations at an offshore station in southern Lake Michigan. J. Great Lakes Res. 7: 290-298.