Environ. Sci. Technol. 1983, 17,357-361
Registry No. PAN, 2278-22-0; CH&OOy, 36709-10-1.
Literature Cited Stephens,E. R. In “Advancesin Environmental Sciences”; Pitts, J. N., Metcalf, R. L., Eds.; Wiley-Interscience: New York, 1969; Vol. 1, pp 119-146. Darley, E. F.; Kettner, K. A.; Stephens,E. R. Anal. Chem. 1963, 35, 589-591. Niebcer, H.; van Ham, J. Atmos. Environ. 1976,10, 115-120. Penkett, S.; Sand&, F. J.;Lovelock,J. E. Atmos. Environ. 1975, 9, 139-140.
(14) Gay, B. W., Jr.; Noonan, R. C.; Bufalini, J. J.; Hanst, P. L., Environ. Sci. Technol. 1976, 10, 82-85. (15) Willner. H. Z. Anorg. A l k . Chem. 1981. 481. 117-125. (16) Shimanouchi, T. Phis. Chem. Ref. Data 1977, 6 ,
993-1102. (17) Siebert,H. “Anwendungen der Schwingungsspektroskopie in der Anorganischen Chemie”;Springer Verlag: Berlin, 1966; p 96. (18) Siebert,H. “Anwendungender Schwingungsspektroskopie in der Anorganischen Chemie”; Springer Verlag: Berlin, 1966; p 93.
(19) Stephens,E. R. Statewide Air Pollution Research Center, University of California,Riverside,personal communication,
Bruckmann, P.; Mulder, W. Schriftenr.Landesanst. Immissionsschutz Landes Nordrhein-Westfalen, Essen 1979,
1979. (20) Milligan, D. E.; Jacox, M. E. J. Chem. Phys. 1964, 42,
47, 30-41.
Stephens, E. R. Anal. Chem. 1964,36, 928-929. Nicksic, S. W.; Harkins, J.; Mueller, P. K. Atmos. Environ.
3032-3036. (21) Dubost, H. Chem. Phys. 1976,12, 139-151. (22) Ohkubo, K.; Sato, H. Bull. Chem. SOC.Jpn. 1979, 52,
1967, I , 11-18.
Penkett, S. A.; Sandalls, F. J.; Jones, B. M. R. In “Ozon und Begleitsubstanzen im photochemischen Smog”;VDI Report, Diisseldorf, 1977, pp 47-54. Varetti, E. L.; Pimentel, G. C. Spectrochim.Acta 1974,30A, 1069-1072. Adamson, P.; Gunthard, H. H. Spectrochim. Acta 1980, 36A, 473-475. Hendry, D. G.; Kenley, R. A. J. Am. Chem. SOC.1977,99, 3198-3199. Cox, R. A.; Roffey, M. J. Environ. Sci. Technol. 1977, 11, 900-906. Kacmarek, A. J.; Solomon,I. J.; Lustig, M. J. Inorg. Nucl. Chem. 1978,40, 574-576.
1525-1526. (23) Jacox, M. E. Chem. Phys. 1981, 59, 213-230. (24) Viossat, V.; Chamboux, J. Bull. SOC.Chim. Fr. 1972, 1699-1702. (25) Bennett, J. E. J. Am. Chem. SOC.1973, 95, 4008-4010. (26) Weaver, J.; Meagher, J.; Shortridge, R.; Heicklen, J. J. Photochem. 1975,4,341-360. Received for review October 22, 1982. Accepted February 28, 1983. H.W. acknowledges generous support by the Fonds der Chemie.
Seasonal Variation of Cadmium Toxicity toward the Alga Selenastrum capricornutum Printz in Two Lakes with Different Humus Content Marit Laegreid,* Joroif AlstadVtDag Klaveness,’ and Hans Martln Selpt University of Oslo, Department of Chemistry, Blindern, Oslo 3, Norway, and university of Oslo, Department of Limnology, Blindern, Oslo 3, Norway
w The alga Selenastrum capricornutum Printz is used to investigate the potential of natural lake water to reduce cadmium toxicity. The two lakes involved differ in trophic status and in concentration and composition of dissolved organic matter, one being a typical dystrophic bog lake, the other a less humus influenced, eutrophic lake. In the dystrophic lake, the toxic effect is determined mainly by the free cadmium activity. In the eutrophic, less humus influenced lake, however, the toxic effect shows considerable seasonal variations with a toxicity far exceeding what would be expected according to the estimated free ion activity during summer. It is hypothesized that qualitative changes in the composition of the dissolved organic matter during the production period are responsible for this effect. Introduction The availability of metals to living organisms depends on the organism in question, on the speciation of the metals, i.e., if they exist as free ions, as complexes with organic or inorganic ligands or adsorbed to particles, and also on the presence of other anions and cations. Most natural waters have shown a certain capacity to ameliorate metal toxicity toward living organisms. The complexation capacity depends on concentration and composition of Department of Chemistry.
* Department of Limnology. 0013-936X/83/0917-0357$01.50/0
inorganic and organic components in the water, which are made available and regulated through physical, chemical, and biological processes in the catchment area and in the lake itself. A number of authors have proposed that mainly free metal ions are toxic to phytoplankton and that all compounds able to reduce the free ion activity thus reduce the metal toxicity. This has been demonstrated for copper in synthetic seawater by using the chelator tris(hydroxymethy1)aminomethane in the concentration range 1-10 mM (1,2) and ethylenediaminetetraacetic acid (EDTA) in the concentration 0.1 mM (2). It has also been shown for copper in natural seawater ( 3 ) . Several studies of natural freshwater containing large amounts of humic matter have also been carried out. It has been found that increasing humic content causes decreasing metal toxicity toward phytoplankton (4-8). Sunda and Lewis (5) also showed that for copper toxicity in the alga Pavlova lutheri, by measuring the copper ion activity with an ion-selective electrode, the toxicity was a function of free copper activity. Complexation capacity in most natural waters seems associated mainly with dissolved organic matter (9, 10). Dissolved organic matter in lakes is derived from two main sources: allochthonous, i.e., derived from the catchment area; autochthonous, i.e., produced in the lake itself. The concentration and composition of dissolved organic matter may both vary between different lakes and vary seasonally in a single lake, as a result of variation in runoff
0 1983 American Chemical Society
Environ. Sci. Technol., Vol. 17,No. 6, 1983 357
Table I. Mean Composition and Ionic Strength (I)in Lake Lille Bakketjern and Lake Gjersjben Lille Bakketjern Gjersjden 1973' 1975-78b 1978c 1980d Ca, mequiv/L Mg, mequiviL
Na, mequiv/L K, mequiv/L C1, mequiv/L SO,, mequiv/L WCO, mequiv/Il
0.060 0.030 0.035 0.016 0.032 0.150
0.061 0.035 0.039 0.018 0.034 0.189
0
0
0.80
0.22 0.34 0.05
0.37 0.40 0.55
0.83 0.23 0.28 0.06 0.30 0.37 0.53
4.4 6.9-10.4 PH I = 'iZCCiZi~ 3.2 X 1.9 x 10-3 mg of DOCiL 18-25 6-8 ' Data taken from ref 17. Data taken from ref 18. Data taken from ref 16. Data measured by Norwegian Institute for Water Research (NIVR).
from the catchment and the production of dissolved organic matter in the lake itself. The latter is of particular importance in eutrophic lakes. It has been shown that algae, and especially blue-green algae, are able to release organic molecules that may form complexes with metal ions (11-13). Clear-water lakes and lakes with moderate concentrations of humic matter have not received the same attention as more typically humic lakes. Gachter (14), using the metals Hg, Cu, Cd, Pb, and Zn, and Steemann-Nielsen and Bruun-Laursen (15), using Cu, studied the seasonal variation in metal toxicity in moderately humic lakes. Their studies were carried out by adding metals to natural phytoplankton populations under the conditions prevailing at the time of sampling. Both studies show an increasing toxicity during the production period. The authors assume this to be caused by a change in the phytoplankton population toward more vulnerable phytoplankton species. The aims of the present work are, by using one test organism and the toxic metal cadmium, to study the differences in moderating metal toxicity by a highly humic lake system and a less humic, eutrophic one. As there are metalimnetic blooms of the blue-green alga Oscillatoria agardhii in the latter during summer (16), a seasonal variation may occur due to release of metal-binding compounds of algal origin.
Description of the S t u d y Areas Lake Lille Bakketjern, situated in the Romerike district about 40 km north of Oslo, is a small dystrophic bog lake that has previously been subjected to several studies (17, 18). It is surrounded by a quaking bog of Sphagnum moss, with Andromeda1 Oxycoccus vegetation. The lake is meromictic with surface area 0.0029 km2 and maximum depth 10.3 m. Lake Gjersjoen, situated 20 km south of Oslo, is a eutrophic, moderately humus influenced lake. It is situated below the postglacial marine limit, and the bedrock consists of Precambrian gneisses and granites. It is a dimictic lake with surface area 2.7 km2and maximum depth 68 m; 15% of the catchment is arable land on quaternary marine deposits, and the rest is predominantly coniferous forest on sparse podzolic soils. For a closer description of the lake see ref 16. The lakes differ both in ionic composition and in concentration of dissolved organic matter (see Table I). The ionic strength is much higher in Lake Gjersjoen, while the dissolved organic matter is more than 3 times greater in Lake Lille Bakketjern than in Lake Gjersjoen. The dissolved organic matter in the two lakes differs markedly in 358
Environ. Sci. Technol., Vol. 17, No. 6, 1983
A
uI
C
h
w
L-d vo vt
Flgure 1. Profiles of elution through Sephadex G-25 fine. Eluting solution is 0.02 M KNQ, at pH 7.00 $: 0.05: (A) elution of blue dextran dissolved in 1 M KNO, (this gives V , and V,, respectively); (B) elution profile of water from Lake Lille Bakketjern containing about 25 mg of DOC/L; (volume of application is 5 mL of 10 times concentrated water); (C)elution profile of water from Lake Gjersjaen containing about 6 mg of DOWL (volume of application is 5 mL of 20 times concentrated water).
molecular size, aromaticity, and hydrophobicity. The dissolved organic matter from Lake Lille Bakketjern seems to be more uniform in structure, giving only one peak on elution through Sephadex G-25, with higher molecular weight than the three peaks obtained for Lake Gjersjoen (Figure 1). Measurements of phenol-reactive (aromatic) groups by the method of de Haan (19) gave about twice as much phenol-reactive groups per unit dissolved organic carbon in Lake Lille Bakketjern as in Lake Gjersjoen (20). The acidity of the two lakes is also quite different. pH in Lake Lille Bakketjern is about 4.4 while Lake Gjersjoen is neutral during winter time, and during the production period pH varies from 7 to more than 10.
Materials and Methods All laboratory glassware were thoroughly washed before use. The washing procedure involved boiling in sodium carbonate solution followed by rinsing in water and then standing for 24 h in 20% nitric acid before final rinsing six times in deionized, distilled water. The biotest experiments were run in 500-mL Erlenmeyer flasks. A set of 16 flasks was used for biotesting with cadmium. Since the same flasks were always used with the same metal concentration, the flasks were washed as described above only once; after use they were rinsed several times in distilled water, dried, and covered with A1 foil. Water samples were taken with a 2-L Plexiglass sampler from the pelagial zone of the lakes, at a depth of 4 m in Lake Gjersjoen and 1 m in Lake Lille Bakketjern. The water was brought to the laboratory in a 25-L polyethylene carboy and immediately filtered through prewashed Whatman GF/C glass fiber filters. Subsamples for chemical characterization of dissolved organic matter were adjusted to pH 7.0 before analysis. Because of the variation in pH in Lake Gjersjoen during the production period, the water was bubbled with clean air for equilibration with COz. The tests were therefore run at the equilibrium pH (around 7.8) in Lake Gjersj~len water. The pH of samples from Lake Lille Rakketjern was increased from 4.4 to 7.5 with 1 N sodium hydroxide and then equilibrated with air before biotesting.
Although the pH adjustment would alter organic chelation, it was done in order to keep experimental conditions comparable from one experiment to the other. The green alga Selenastrum capricornutum, used as a test organism in these experiments, belongs to the group of ubiquitous species that have a wide tolerance toward environmental conditions (21). Precultures of algae were run in sterilized water from the same lakes as the experiments, only with the nutrient salts K2HP04and NaN03 added. Algae for the experiments were always taken from a culture in exponential growth at a density of about lo6 cells/mL. Selenastrum cells were transferred to the test solution to a final concentration of about 2000 cells/mL. Equilibrated lake water (250 mL) was transferred to the 500-mL Erlenmeyer flasks, and the nutrients K,HPO4 and NaN03 were added to a concentration of 0.2 and 2 mg/L, respectively. The algae were exposed to total cadmium concentrations ranging from 3.6 X to 3.6 X lo4 M. Cadmium dilutions were made from a stock solution of 1 g/L cadmium as CdS04. The experiments were run in duplicate, with two flasks run as a blank with no metal addition and with six to seven different metal concentrations. In one instance we used five replicates for four metal concentrations. The standard deviations were in all cases about 2 9%. Lake water and metal were allowed to equilibrate for 24 h before the algae were added. The experiments were run at 17 OC with a 12/12 hours light/dark cycle. The tests were always started at the same hour of day. After 24 h of cadmium exposure, the flasks were thoroughly shaken, and 50 mL of the suspension was transferred to 50-mL glass-stoppered bottles. A sterilized 0.2 pCi/mL [14C]HC03soln. (0.5 mL) was added, and the flasks were placed for 2 h in an incubator at 20 OC and 170-peinsteins light intensity. The solutions were then filtered through 0.45-pm Millipore filter and the filters allowed to dry before addition of 10 mL of scintillation solution. The toxic response was measured as the reduction of 14Cuptake relative to the blank. Free cadmium ion activity was measured with an ionselective electrode (Orion Model 94-48A) coupled to a single-junction Ag/AgCl reference electrode (Orion Model 90-01). pH was measured with a combination glass electrode (Beckman Model 39012). Measurements were performed under constant pH and temperature. The temperature was thermostatically controlled with a circulating water bath at 20 f 0.5 “C. Both standard solutions and tests were run in 0.01 M KN03. Before each experiment the electrode was polished to give reproducible and rapid response. A standard curve with the same metal concentration as the test was taken each time. Test solution with lake water, metal, and potassium nitrate was put into 100-mL polyethylene flasks and allowed to equilibrate for 24 h. These solutions were divided into two portions, one for electrode equilibration and one for potential reading. Reading was done when the potential was stable to within f O . 1 mV for 1min. Provided the electrode was adequately polished and given the necessary equilibration time, the response was reproducible to within f0.5 mV. The electrode responded according to the theoretical Nernstian slope down to a total cadmium concentration of lo4 M, whereafter the slope decreased. The ion-selective electrode measurements gave good results on water from Lake Lille Bakketjern but did not yield reliable results on water from Lake Gjersjeren. Three types of biotests were performed to estimate the free cadmium ion concentration causing a certain toxic effect in Lake Gjersjeren water, Le., the method suggested
by Borgmann (22),tests on ultrafiltered water samples, and tests in the artifical medium “Fraquil”. In the ultrafiltration experiments, glass-fiber-filtered water samples were filtered through an Amicon Diaflo UM-05 ultrafiltration membrane, with a cutoff limit for molecules with a molecular weight of 500. The biotest experiments were performed on the samles passing through this filter. The method of Borgmann is based on the assumption that the difference in total metal concentration causing a certain toxic effect in lake water with a known ligand addition and the total metal concentration causing the same effect in lake water without ligand addition gives the concentration of metal bound to the added ligand. Thus the free metal concentration causing the toxic effect may be calculated from known stability constants. In our experiments we used the ligand nitrilotriacetic acid (NTA). For Lake Lille Bakketjern nearly the same toxic effects of cadmium were observed with NTA concentrations of 1.0 x lo4 and 2.5 X lo4 M. With the former concentration of NTA the estimated free cadmium concentration agreed quite well with the value obtained with the ion-selective electrode. An NTA concentration of 1.0 X lo4 M was therefore used also for Lake Gjersjeren. The artificial medium “Fraquil” is a freshwater version of the “Aquil” medium designed for metal studies (23), where the seawater salts are substituted by the main constituents as in WC medium (24). Biotest experiments were performed in this medium, omitting EDTA and copper, and with citric acid at concentrations of 1 X 6X and 1X lo4 M as the only organic ligand added. Results Lake Lille Bakketjern. Biotest experiments on water from Lake Lille Bakketjern were run for samples taken in March, May, June, and Sept 1981. Concentrations of free cadmium ions were measured with an ion-selective electrode. Concentrations of dissolved organic carbon, analyzed on a CHN analyzer, were about 25 mg/L in March and about 18 mg/L during May, June, and Sept. The per cent inhibition of 14C uptake as a function of total cadmium addition is shown in Figure 2A. The total cadmium concentration causing 50% inhibition of 14Cuptake varies in the range (7.2 X 10-7)-(1.5 X lo*) M. If, on the other hand, the per cent inhibition of 14C uptake is plotted as a function of measured free cadmium concentration, the points fall nicely on a single curve as shown in Figure 2B, indicating the toxicity response to be a function of free metal ion concentration. This is in agreement with the results obtained by others for typical humic localities (cf. Introduction). In Lake Lille Bakketjern, the free cadmium concentration causing 50% inhibition of 14Cuptake falls in the range (3-4) X lo-’ M. Lake Gjersjeren. The biotest experiments with Lake Gjersjaen water were run regularly (usually monthly) during a 3-year period. The results obtained do not conform to the same simple pattern found for typical humic localities, here exemplified by Lake Lille Bakketjern. Toxicity response curves from May and Sept 1981 are shown in Figure 3. As this figure shows, the algae are almost unaffected by a total cadmium concentration of 3.6 X M in May, whereas in Sept a toxicity response is observed even at this low cadmium concentration. A plot of total cadmium concentration causing 50% inhibition of 14Cuptake vs. time (Figure 4) indicates an increasing cadmium toxicity during summer. This increased toxicity lasts until the lake turnover in the autumn. These results are in accordance with those obtained by Gachter (14) and Steemann-Nielsen and Bruun-Laursen (15). Since our Environ. Sci. Technol., Vol. 17, No. 6. 1983
359
P
A
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80
10OlO8
‘.-‘8.t
0.
U
1
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-
Free cadmium concentration (MI Flgure 2. Plot of the per cent inhibition of ‘“Cuptake vs. concentration of cadmium in water from Lake Lilie Bakketjern (0,03/03/81; A,
05/13/81; 0,06/30/81; W, 09/12/81): (A) per cent inhibition vs. total cadmium concentration; (6) per cent inhibition vs. measured free cadmium concentration.
!OI 0
sL 20
f 801 U
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I
107 10.6 Total cadmium concentratlon(M)
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Figure 3. Toxicity response curves from Lake Gjersjoen at two different dates: 0 , 05/18/81; A 09/28/81.
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l o s ‘ M I J 1J 1 A l s l o l N l D J ~ F ~ M I A I M1JI JI A I S I O L N I D / J I F 1MtAlMl J 1 J ‘ A I s1olN I980 1981 1982
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Figure 4. Variation in total concentration of cadmium causing 50% inhibition of ‘“Cuptake in Lake Gjersjoen. The shaded area Indicates the estimated free concentration of cadmlum causing 50% inhibition.
experiments were performed under constant experimental conditions with respect to pH, temperature, algal species and density, light/dark-cycles, etc., the response variation cannot be explained by a change in phytoplankton population toward more sensitive species in the summer, as proposed by the above-mentioned investigators. The explanation is more likely chemical changes in the lake water. The variations in cadmium toxicity show, however, no 360
10
(MI
significant correlation with dissolved organic carbon. Also, Gchter (14) was not able to explain his observations by variations in pH value, concentration of calcium, dissolved organic nitrogen, or allochthonous debris. The question now is whether (3-4) X M of free cadmium ions also causes 50% inhibition for water from Lake Gjersjoen as found for water from Lake Lille Bakketjern. As already mentioned, reliable results were not obtained when employing the ion-selective electrode in Lake Gjersjoen water. We have therefore tried three different experimental approaches in order to evaluate the free cadmium ion concentration. In the ultrafiltration experiment, gel filtration of the dissolved organic matter retained by the filter showed that the lower molecular weight fraction in Lake Gjersjoen passed this filter. This constituted only 15% (1 mg of DOC/L) of the total dissolved organic matter and should only bind a small fraction of cadmium. Biotest experiments performed on ultrafiltered water samples taken during winter time gave the toxic response of 50% inhibition at a total cadmium concentration of about 8 X M. Toxicity measurements after the method of Borgmann were performed on water samples from Oct and Nov 1981. The calculated free cadmium ion concentration causing 50% inhibition in these experiments was about 8 X
M.
d
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IO5
Flgure 5. Biotest experiments at varying ionic strength in Lake Lille Bakketjern water: 0 , without ionic addition; W, with addition of CaCI,, MgS04, and NaNO, to the same strength as in Lake Gjersjoen.
B
f 80-
10-6
Total cadmlum concentration
Total cadmlum concentration ( M )
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Environ. Sci. Technol., Voi. 17, No. 6, 1983
In the artificial “Fraquil” medium the toxicity measurements were performed with 10-7-10-6 M citric acid as the only organic ligand present. The ionic strength of this medium corresponds to that of Lake Gjersjoen. In this case the calculated free cadmium concentration causing 50% inhibition falls in the range (8-10) X M. The results from these experimental approaches indicate that a free cadmium ion concentration in the range (8-10) X M would cause a 50% inhibition of 14Cuptake in Lake Gjersjoen. The difference between the two lakes with respect to the free metal ion concentration causing 50% inhibition may partially be explained by the difference in their salinity. Addition of CaCl,, MgSO,, and NaN03 to samples from Lake Lille Bakketjern to the same ionic strength as that found for Lake Gjersjoen results in a displacement of the toxicity response curve toward higher cadmium tolerance (Figure 5). As a reasonable estimate of the free cadmium ion concentration causing 50% inhibition for samples from Lake M, the curve of 50% Gjersj~lenseems to be (8-10) x inhibition vs. time ought to lie above the shaded area shown in Figure 4,indicating cadmium binding, or in the shaded area, indicating no cadmium binding. One would not expect the 50% inhibition to fall below this area. Most of the year the curve fluctuates around the area of no cadmium binding. During the months June-Sept,
however, it falls far below, indicating a cadmium toxicity far exceeding that expected if no cadmium binding occurs (Figure 4). The toxic response also exceeds the value found in Lake Lille Bakketjern. The presence of trace elements in Lake Gjersjraen were measured by inductively coupled emission spectroscopy (ICP) on unfiltered, acidified water samples from June 2, Aug 16, and Nov 17, 1982. Trace elements like copper, zinc, lead, cadmium, and nickel were all below detection limit (5 ppb for Cu and Zn, 25 ppb for Pb, 10 ppb for Cd and Ni). The concentration of cadmium was also .below the detection limit of 0.5 ppb measured by neutron activation analysis.
During the phytoplankton bloom, one would expect increased concentrations of low molecular weight organic molecules as a result of extracellular production, autolysis, and decomposition. In addition, the increased solar radiation during summer months at 60’ N may add to the breakdown of polymer organic compounds into simpler species. An increased cadmium uptake mediated by these organic molecules may serve as a hypothesis for the increase in toxicity during summer. Registry No. Cd, 7440-43-9.
Literature Cited Sunda, W. G.; Guillard, R. R. L. J. Mar. Res. 1976, 34, 511-529. Anderson, M. A.; Morel, F. M. Limnol. Oceanogr. 1978,23, 283-295. Srna, R. F.; Garrett, K. S.; Miller, S. M.; Thum, A. B. Enuiron. Sci. Technol. 1980, 14, 1482-1487. Giichter, R.; Davis, J. S.; Mares, A. Enuiron. Sci. Technol. 1978. 12. 1416-1422. Sunda, W,G.; Lewis, J. A. M. Limnol. Oceanogr. 1978,23, 870-876. Gjessing, E. T. Arch. Hydrobiol. 1980, 91, 144-149. Toledo, A. P. P.; Tundisi, J. G.; D’Aquino, V. A. Hydrobiologia 1980, 71, 261-263. Sedlacek, J.; Kiillqvist, T.; Gjessing, E. T. In “Aquatic and Terrestrial Humic Materials”; Christman, R. F., Gjessing, E. T., Eds.; Ann Arbor, Science: Ann Arbor, MI, 1983; pp 495-516. Sunda, W. G.; Hanson, P. J. In ”Chemical Modeling in Aqueous Systems”; Jenne, E. A,, Ed.; American Chemical Society: Washington, D.C., 1979; ACS Symp. Ser. 93, pp 147-180. Blutstein, H.; Shaw, R. F. Enuiron. Sci. Technol. 1981,15, 1100-1102. Simpson, F. B.; Neilands, J. B. J. Phycol. 1976,12,44-48. Armstrong, J. E.; Van Baalen, C. J. J.Gen. Microbiol. 1979, 111, 253-262. McKnight, D. M.; Morel, F. M. M.; Limnol. Oceanogr. 1980, 25, 62-71. Gachter, R. Schweiz. 2.Hydrol. 1976, 38, 97-120. Steemann-Nielsen, E.; Bruun-Laursen, H. Oikos 1976,27, 239-242. Faafeng, B.; Nilsen, J. P. Verh. Int. Verein. Limnol. 1981, 21, 412-424. Hongue, D. Schweiz. 2.Hydrol. 1980,42, 171-195. Lavh~iden,F. Thesis, University of Oslo, in preparation. Haan, H. de. In “Humic Substances. Their structure and function in the Biosphere”; Povoledo, D.; Golterman, H. L., Eds.; Wageningen, 1975; pp 53-62. Laegreid, M. Thesis, University of Oslo, in preparation. Rodhe, W. Mitt. I n t . Verein. Limnol. 1978, 21, 7-20. Borgmann, U. Can. J.Fish Aquat. Sci. 1981,38,999-1002. Morel, R. M. M.; Reuter, J. G.; Anderson, D. M.; Guillard, R. R. L. J. Phytol. 1979,15,135-141. Guillard, R. R. L.; Lorenzen, C. J. J. Phycol. 1972, 8, 318-323. Harriss, R. C. Biol. Conseru. 1971, 3, 279-283. Poldoski, J. E. Enuiron. Sci. Technol. 1979, 13, 701-706. Laube, V. M.; McKenzie, C. N.; Kushner, D. J. Can. J. Microbiol. 1980, 26, 1300-1311. Giesy, J. P., Jr.; Levenee, G. T.; Williams, D. R. Water Res. 1977, 11, 1013-1020. Sedlacek, J.; Kallqvist, T.; Gjessing, E. 1982. Norwegian Institute for Water Research, 1. Nat. Report; Oslo, Norway: NTNFs utvalg for drikkevannsforskning, Drikkevannsrapport May 1982.
Discussion In agreement with earlier investigations, we find that the toxicity response by metal additions in a simple humic lake system like Lille Bakketjern is a function of metalorganic mr)tter interactions: the toxic response corresponds to a certain free metal Concentration, and metal-humic binding does not contribute to the toxicity. In a moderately humic influenced, eutrophic lake like Lake Gjersjraen, an increasing metal toxicity is observed dufing the growth season. Since experimental conditions were kept constant, the reason for this increased toxicity must be due to chemical changes in the lake water. The reason seems not to be a reduction in metal binding since the toxicity exceeds that expected if the total metal addition is available as free metal. Working with experimental “blanks” does not eliminate the possibility of synergistic effects with trace metals originally present in the lake water. Although this possibility could not be completely eliminated, it is not a likely explanation since the concentrations of other trace elements in the lake water are very low. Furthermore, a large fraction of these metals may be bound to particulate matter and thus become removed upon filtration. Also, the concentration of particulate organic matter in the form of algae and detritus are at its maximum during summer. For several years the usual working hypothesis has been that only the ionic, but not the complexed metal, is toxic ( I ) . Variations in toxic response not explainable in terms of changes in the free metal concentration have long been known for mercury: several organomercurials are 100 times as toxic as inorganic mercuric chloride toward phytoplankton (25). A similar behavior is found for cadmium and the ligand diethyldithiocarbamatetoward zooplankton (26). Laube et al. (27)found that NTA increased the toxic effect of copper, cadmium, and lead toward a blue-green alga but not toward a green alga. Experiments done in this laboratory with the chelators NTA and citric acid and the same test alga indicate that the toxicity is a function of not just free metal ion concentration but also of type and concentration of ligand used (20). Giesy et al. (28))working with zooplankton, and Sedlacek et al. (29))working with phytoplankton, found by doing metal-uptake experiments that the smallest molecular weight fractions of humic substances caused a somewhat increased metal uptake. Our observations, supported by the evidence referred to above, indicate that the assumed close connection between free metal ion activity and toxicity is not generally valid. Other mechanisms may also be of importance. It appears that certain low molecular weight organics may increase the metal uptake and thus increase the toxicity.
Received for review October 26, 1982. Accepted February 28, 1983.
Environ. Scl. Technol., Vol. 17, No. 6, 1983 381
