Sensitivity of estuarine phytoplankton to hexavalent chromium

Estuarine, Coastal and Shelf Science (1983) 17, 181-187

Sensitivity of Estuarine Phytoplankton Hexavalent Chromiuma

Bruce E. Frey, G. Fritz F. Small

Riedel,

to

Ann E. Bass and Lawrence

School of Oceanography, Oregon State University, Corvallis, Oregon 97331, U.S.A. Received 10 August 1980 and in revised form IS October 1982

Keywords:

chromium;

estuarine flora; salinity variations;

Oregon coast

Experiments were conducted in which we tested the effects of hexavalent chromium on both naturalassemblages of phytoplanktonandculturesof Thalassiosira pseudonana clone 3H. Water was collected from various locations in Yaquina Bay, Oregon, with salinities ranging from 32.5% to 0’03%~ A variety of nutrient regimes were tested by adding major nutrients and micro-nutrients, and/or stripping micro-nutrients with activated carbon. In one high salinity experiment, chromium was stimulatory, as were micro-nutrient additions. In other high and medium salinity experiments, chromium was neither stimulatory nor inhibitory, at levels up to 1.9 l-t mole 1-l Cr. There was, however, slight inhibition of growth at 19.0 u moleI-1 Cr, due specificallyto inhibition of Skeletonema costatum. In our freshwater experiments, chromium was very inhibitory at 1.9 lt mole 1-r Cr, and slightly inhibitory at 0.19 p mole 1-i Cr. Species inhibited by chromium were Surirella ova&a, Detonuia confervacea, and Cyclotella sp. Experiments were conducted with T. pseudonana grown over a wide range of salinities. Chromium was found to be very inhibitory in freshwater and became progressively less toxic as the salinity increased. Most inhibition was neutralized by a salinity of 2 1%0. Introduction

Chromium is an important trace constituent of industrial and domestic waste, yet there exists little information regarding its effects on natural freshwater, estuarine, or marine phytoplankton. Of the commonly occurring forms of chromium (III and VI), the hexavalent form seemsto be the most toxic (Towill ecal., 1978). Almost all the work dealing with the effects of hexavalent chromium on algae hasbeen conducted in freshwater. Patrick et af. (1975) found in freshwater that chromium levels of about 1.9 u mole 1-I Cr inhibited diatom growth in a mixed population, and at about 7 ‘6 u mole l-1 Cr, diatoms were completely replaced by blue-green algae. In cultures of the freshwater algal speciesNitzchia palea and Chlorella pyrenoidosa, Wium-Andersen (1974) found severe inhibition of growth at 5-7 ymole 1-l Cr, moderate inhibition at 2.9 u mole 1-l Cr, and slight inhibition at 1.O l.t mole 1-l Cr. Nollendorf et al. (1972) found inhibition of the growth of Chloretta sp. at chromium concentrations greater than 9.5 p mole 1-i Cr. Selewstrum capicurnutum was severely inhibited by chromium concentrations of 26.6 u mole 1-l Cr (Garton , 1973). Clearly, hexavalent chromium is toxic at certain levels in freshwater, and the toxicity varies from speciesto species. ‘This work was supported by the U.S. Environmental Protection Agency (R805282

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Little work has been performed on the effects of hexavalent chromium on marine algae. Hollibaugh et al. (1980) showed no chromium toxicity to natural populations of marine phytoplankton or unialgal cultures of the marine species Thalassiosira aestivalis, at a concentration of 1 .O p mole 1-l Cr. Toxicity of hexavalent chromium (and most other toxic substances) to algae has not been examined in the estuarine environment. In light of the possibly divergent results obtained from work in freshwater and seawater, the effects of chromium on natural phytoplankton populations in estuarine waters appears of particular interest. Materials

and methods

Seven long-term experiments using natural phytoplankton populations, and two unialgal culture experiments using Thalassiosira pseudonana clone 3H, were conducted. T. pseudonana clone 3H was originally isolated from Mariches Bay, New York (Guillard & Ryther, 1962). For the natural population experiments, water was collected from three stations on Yaquina Bay, Oregon, at seven times during the years 1977-1979. The water was pumped from a depth of 0.5 m and passed through a spun polypropylene Framweb@ pre-filter and a Pall DE@ 3 pm pore-size cartridge filter. Water thus filtered was pumped into two 150-gallon polyethylene tanks. To strip out some trace organics and trace metals, Darco G-60 activated carbon (1 g 1-i) was added to one of the tanks. The water was stored for two days, during which period it was vigorously stirred several times. At the end of the two-day period, all water, both carbon-treated and non-treated, was filtered directly into new, rinsed, lo-litre polyethylene Cubitainer@ growth containers through a Framweb@ pre-filter and a Pall Ultipore @ 0.45 pm pore-size cartridge filter. Treatment additions consisted of various combinations of chromium (added as sodium dichromate to give 1.9, 0.19, and 0.019 u mole 1-i Cr), the major nutrients (N, P, Si), trace metals (Fe, Cu, Zn, Co, Mn, MO), and vitamins (thiamin, biotin, B,,). Nutrients were added as l/10 strength medium f (Guillard & Ryther, 1962). Incubation was carried out by suspending the growth containers in a large, outdoor, temperature-controlled water bath, kept within 1 “C of the collection temperature under the natural die1 light-dark cycle. The growth containers were inoculated with unfiltered water containing natural phytoplankton from Yaquina Bay. Water for the inoculum was collected at the same location and depth as the experimental water, during the high tide preceding inoculation. Ten milliliters of inoculum were added by repipet to each IO-litre growth container. There were three replicates of each treatment, except where noted in the results. Samples were collected daily between 1030 and 1200 hours from each growth container. They were kept at the temperature of the incubation tank until analysed. Growth was monitored by measuring in vivo flourescence and extracted chlorophyll a through time. Fluorescence measurements were carried out with a Turner Designs@ fluorometer, equipped as recommended by the manufacturer. To enhance the precision of in vivo fluorescence measurements, samples were kept in the dark at the incubation temperature for an hour prior to measurement. Extracted chlorophyll a measurements were made according to the fluorescence technique of Yentsch & Menzel (1963). At the peak of phytoplankton growth, samples were taken for microscopic enumeration. These samples were preserved in Lugol’s solution, and at least 200 cells were counted in each sample. Unialgal culture experiments with Thalassiosira pseudonana clone 3H were conducted to test in detail the effect of salinity on the toxicity of hexavalent chromium to phyto-

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plankton. T. pseudonana clone 3H was selected for this study because it grows well over the full range of salinities to be tested (Guillard & Ryther, 1962). Cultures were grown in 500 ml polycarbonate Erlenmyer flasks at 20 “C, with 250 microeinsteins m-2 set-3 continuous fluorescent illumination. Water for the T. pseudonana experiments was collected from the freshwater end of Yaquina Bay (0.03%0) and from the high salinity end of the bay (32.5%0). All water was filtered (0.45 mp pore-size) and autoclaved. Major nutrients (nitrate, phosphate, and silicate) were added at l/IO strength medium f concentrations. A range of salinities was developed by mixing low and high salinity water. In our initial experiment, the toxicity of three concentrations of chromium to T. pseudonana (zero, 0.19, and 1.9 p mole 1-i Cr, added as sodium dichromate) was examined over a range of salinities from 0.03%0 to 32.5%0. In the second experiment chromium concentrations of zero, 0.38, and 3.8 p mole 1-l Cr were tested over a greatly reduced salinity range of 0.03960 to 2.11%0. Cultures were monitored daily with measurements of in vivo fluorescence. In addition to bioassays, we performed Cr VI analyses to determine if the hexavalent chromium additions remained in the solution and if the additions remained hexavalent. Chromium VI additions remaining in both freshwater and seawater after two-month incubations in lo-litre cubitainers was determined. Sodium dichromate (1.9 ~1mole 1-l Cr) was added to filtered (0.45 mp) seawater and freshwater. Chromium VI was concentrated and separated from chromium III by use of an anion exchange resin (AGI-X4) and reactive ion exchange elution (Pankow & Janauer, 1974), with 1% W/V hydroxylamine*HCl in 12.5% V/V HNO, as the elutant. Analysis was carried out on a Varian AA-5@’ atomic absorption spectrophotometer, with a neutral flame being used to avoid potential interferences. We were able to recover nearly all the additions as chromium VI after two months. The nominal initial addition was 1.9 l.r mole 1-i Cr VI and we recovered 1.8 f 0.2 l.r mole 1-l Cr VI in seawater (3 replicates), and 1.6kO.2 p mole 1-l Cr VI in freshwater (4 replicates). Thus there was little if any, reduction of chromium VI to chromium III, or loss of chromium by adsorption to containers walls in our experimental seawater and freshwater. These results are consistant with the findings of Cutshall et al. (1966), in which chromium VI introduced into the ocean was found to remain in the hexavalent state. As a further test of possible losses of chromium VI to growth container walls, we tested chromium-51 in filtered (0.45 mp) seawater. Four polyethylene containers were each tiled with 11 of the water, and 1 FCi chromium-51 (added as sodium dichromate) was added to each container. Over a 33-day period, 5-ml aliquots were collected and analysed for chromium-51 on a Nuclear Data ND 128@ multi-channel analyser coupled to a sodiumiodide detector. Maximum loss of chromium to the walls was 2% after 33 days. In freshwater, Schroll (1978) found no evidence of hexavalent chromium-5 1 adsorption in cultures of the algae Chlorella pyrenoidosa. Results

and discussion

Among the seven natural population experiments, four were conducted with high-salinity water (about 32.5%0), two with low-salinity water (0.03 and 0.04%0), and one with intermediate-salinity water (20.4%0). Results from these experiments are shown in Figures 1 and 2. While in vivo fluorescence results are shown in the figures, extracted chlorophyll a measurements (less frequent) showed the same results. Day 0 is when inocula were added, however, since there was a 1 : 1000 dilution of the natural population concentration, fluorescence was not measurable above background levels until a number of cell divisions had taken place. The number of days elapsed until fluorescence became measurable was

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Days of growth Figure 1. Effects of chromium additions and micro-nutrient additions on phytoplankton growth in high salinity water collected in November, 1977. Results in both non-carbontreated and carbon-treated water are shown. Lines join means of two replicates, and vertical bars show ranges. (-Control; . . . . 1.9 pm01 l-1 Cr; -- 0.19 pm01 1-l Cr; - 0.019 pool; -- Fe, trace metals, vitamins.)

a function of initial inoculum density, fraction of viable cells, growth rates, and lags. The X axes of Figures 1 and 2 show days beginning when in orno fluorescence reached levels readable above background in the experimental cultures. In the first high-salinity experiment (November, 1977), one in which cultures were evidently micro-nutrient-limited, the addition of chromium gave a marked enhancement of growth (through reduced apparent lags or increased early growth rates) similar to or greater than the enhancement caused by the addition of iron, other trace metals, and vitamins. Enhancement was greatest at the 0.019 p mole 1-i Cr concentration, and somewhat less at higher chromium concentrations. A similar pattern was shown in the more severely micro-nutrient-limited, carbon-treated, water (Figure 1). The chromium and micro-nutrient treated cultures showed an increased yield of two speciesof the diatom Nifzchia. Among the other high salinity experiments, there was no observable inhibitory or stimulatory effect of added chromium on the developing phytoplankton populations nor was there any effect on the species composition, at concentrations up to 1.9 p mole 1-i Cr. However, in March, 1978, the only time that an unrealistically high 19.0 u mole l-1 Cr wastested, an apparent lag at the 19.0 u mole 1-l Cr level wasobserved. The diatom Skeletonemu costut#mwas eliminated by this concentration, and the apparent lag relative to the control may be due to the elimination of the S. costatum component of the population. In the medium salinity experiment chromium had little or no effect relative to the control. In contrast, in the low salinity experiments the 1.9 u mole 1-l Cr additions either completely eliminated measurable growth of phytoplankton (January, 1979, Figure 2), or greatly reduced the rate of growth (October, 1978, Figure 2) relative to controls. The 6; 19 u mole 1-l Cr additions causedan apparent lag in growth relative to the controls in both of these low-salinity experiments. Speciesinhibited by the chromium additions were

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Figure 2. Effects of chromium additions on phytoplankton growth in two low salinity experiments. Results with and without nutrient additions are shown. Lines join medians of three replicates, and vertical bars show ranges. (-Control; . 1.9 pm01 1-l 0; -- 0.19 pm01 I-1 Cr. In the bottom two parts of the figures there was no growth 1-l Cr; - 0~019pmal at 1 ,9 pm01 1-I Cr.)

Surirella ovata, Cyclotella sp., and Detonula confervacea. Similar results were found when nutrients were added in these experiments (Figure 2). In our initial experiment with T. pseudonana, at the lowest salinity 0.19 p mole l-1 Cr was moderately inhibitory and 1.9 p mole 1-i Cr was severely inhibitory to growth. At all higher salinities (4 ‘0 to 32.5%0) there was no effect at either level of chromium addition. In our second experiment with T. pseudonana, a detailed examination of chromium toxicity over a range of low salinities was made. At the lowest salinity (O-03!&), growth rate and yield in the control was similar to controls at higher salinities, but both 3.8 and 0.38 u

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Figure 3. Effects of chromium of low salinities. (- Control;

additions on growth of Thalassiosira -- 3 .l3 pm01 I-1 Cr.)

psetufonana

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mole 1-r Cr additions inhibited all growth (Figure 3). At the next higher salinity (0.09!%), the high chromium additions still suppressed all growth; however, there was growth at a reduced rate with the low chromium addition. At 0.29%0 salinity, there was still no growth with the high-chromium addition, but there was only slight inhibition of final yield with the low chromium addition. At 1.10960 salinity, the low chromium addition caused no inhibition of growth, and there was slow growth with the high chromium addition. At the highest salinity of this experiment (2~11%) there was no inhibition of growth caused .by

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the low chromium addition, and relatively slight inhibition of growth rate and final yield from the high chromium addition. It is clear from both the natural population experiments and the T. pseudottana experiments that salinity exerts a strong effect on the toxicity of hexavalent chromium. That seawatercan neutralize toxic effects of hexavalent chromium on phytoplankton hasobvious implications for the disposal of chromium in natural waters. The mechanism of this detoxification is not yet clear. While most ionic forms of metals studied to date are cationic (e.g. Mn2+, Cuz+, Cdz+, Pb2+ and HgZ+), hexavalent chromium in water is anionic (its forms being predominantly Cr0,2- and HCrO,-). The chemistry and toxicological mechanismsof chromium-VI can thus be expected to be quite different from that of these other metals. There is a growing body of literature showing that the biological activity of the cationic metals is related to the activity of the free ion, rather than the total analytical concentration including complexed forms (Sunda & Guillard, 1976; Anderson & Morel, 1978; Anderson et al., 1978; Canterford & Canterford, 1980; Anderson & Morel, 1982). Since chromium VI is anionic, it has little affinity for organic ligands. Thus, unlike the cationic metal ions, it will not be regulated and detoxified by organic ligands in the aqueous media, and its mechanism of toxicity at the cell surface or within the cell may be fundamentally distinct from that of the cationic metals. The detoxification of hexavalent chromium by relatively small amounts of seawater is a starting point which might, with further study, give a better understanding of the mechanismsof toxicity of this metal. References Anderson, D. M. & Morel, F. M. M. 1978 Copper sensitivity of Gonyaulax tamarensis. Limnology and Oceanography 23,283-295. Anderson, M. A. & Morel, F. M. M. 1982 The influence of aqueous iron chemistry on the uptake of iron by the coastal diatom Thalassiosira weisspogii. Limnology and Oceanography 27, 789-813. Anderson, M. A., Morel, F. M. M. & Guillard, R. L. L. 1978 Growth limitation of the coastal diatom by low zinc ion activity. Nature 276, 70-71. Canterford, G. S. & Canterford, D. R. 1980 Toxicity of heavy metals to the marine diatom Dirylum brtghtwellii (West) Grunow: Correlation between toxicity and metal speciation. your& of rhe Marine Biological Association of the United Kingdom 60, 227-242. Cutshall, N., Johnson, V. & Osterberg, C. 1966 Chromium-51 in seawater: chemistry. Science, 152 (3719), 202-203. Garton, R. B. 1973 Biological effects of cooling tower blowdown. l?‘afer 69 (129), 284-292. Guillard, R. R. L. & Ryther, J. H. 1962 Studies of marine planktonic diatoms. I. Cyclocella nana Hustedt, and Detonula confervalea (Cleve) Gran. Canadian journal of Microbiology 8,229-239. Hollibaugh, J. T., Seibert, D. L. R. & Thomas, W. H. 1980 A comparison of the acute toxicities of ions of ten heavy metals to phytoplankton from Saanich Inlet, B.C., Canada. Estuarine and Coastal Marine Science 10, 93-105. Nollendorf, A., Pakalne, D. 81 Upitis, V. 1972 Little known trace elements in Chlorella culture. Latvijas PSR ZimitTu Akaderuijas Vestis (Latvian S.S.R.) 7, 33-43. Pankow, J. F. & Januer, G. E. 1974 Analysis for chromium traces in natural waters. Part I. Preconcentration of chromate from ppb levels of aqueous solutions by ion exchange. Anal. Chem. Acra 69,97-104. Patrick, R., Boot, T. & Larson, R. 1975 The role of trace elements in management of nuisance growths. EPA66012-75-008, U.S. Environmental Protection Agency, Corvallis, OR. 250 pp. Schroll, H. 1978 Determination of the adsorption of Cr+S and Cr +3 in an algal culture of Chlorella pyrenoidosa using O-51. Bulletin of Environmental Gmtamkation and Toxicology 20, 721-724. Sunda, W. G. & Guillard, R. L. L. 1976 The relationship between cupric ion activity and the toxicity of Cu to phytoplankton. Journal of Marine Research 34, 51 l-529. Towill, L. E., Shriner, C. R., Drury, J. S., Hammons, A. S. & Holleman, J. W. 1978 Reviews of environmental effects of pollutants: III. Chromium EPA-60011-78-023. U.S. Environmental Protection Agency, Health Effects Research Laboratory, Cincinnati, OH. Wium-Anderson, S. 1974 The effect of chromium on the photosynthesis and growth of diatoms and green algae. Physiologia Plantmum 32, 308-310. Yentsch, C. S. & Menzel, D. W. 1963 A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-Sea Research 10, 221.