Environ. Sci. Technol. 1984, 18, 383-385
Longevity of Algal Inhibition after Chlorination of Estuarine Water James G. Sanders Academy of Natural Sciences, Benedict Estuarine Research Laboratory, Benedict, Maryland 20612
Estuarine water was chlorinated to 10 mgL-l, aged 10-35 days, and then used as a growth medium for three phytoplankton species. Total residual chlorine compounds were undetectable in the chlorinated water; however, two species would not grow even after the water had been aged 35 days. A more resistant species grew in chlorinated water aged 23 or 35 days but would not grow in water aged 10 days. All three species grew well in the same water that had not been chlorinated. The degree of chlorination as well as the aging time was important in determining cell growth. In addition, algal resistance to trace metal (Cu) toxicity was reduced, possibly due to increased activity of the free Cu ion. The quantity of organic carbon oxidized during the degradation of chlorine residuals is sufficient to destroy the Cu binding capacity of estuarine waters.
Introduction Chlorine has been in widespread use for decades as a disinfectant for industrial and sewage effluents and as a biocide in water supplies or for removal of fouling organisms from cooling systems (I). Chlorine and its associated oxidants have historically been considered toxic to marine phytoplankton at the low concentrations found in such effluents (2, 3). In addition to overall inhibition of productivity, there is recent evidence that the effects of such compounds may not be felt equally by all algal species (4, 5). For example, natural phytoplankton assemblages exposed to chronic, low-level chlorination (0.05-0.15 mgL-l) exhibited significant shifts in species dominance from assemblages dominated by centric diatoms to a pennate diatom/microflagellate dominance (6). Therefore, chlorination potentially could alter the species composition of plankton communities as well 89 cause an overall reduction in productivity. Even with the large potential toxicity of chlorine to phytoplankton, however, current philosophy suggests that potential for harm to natural systems is not great because of the rapid disappearance of chlorine-produced oxidants in seawater, the relatively short duration of exposure (Le., the time of passage through a chlorinated cooling system), and the high degree of dilution after the chlorine has been applied (7, 8). This philosophy may be in considerable error, however. A number of toxic chlorination byproducts persist indefinitely; in addition, dilution in surface waters is often restricted. Therefore, an algal cell will have an increased chance of contacting a toxic compound. These points, coupled with earlier studies indicating that chronic chlorination at concentrations of only 1 pgL-l causes significant declines in cell growth (9),suggest that the potential for inhibition is greater than previously considered, especially if toxic compounds produced as a result of chlorination are long-lived. In addition, chlorination of natural waters can increase the activity of toxic metal ions by destroying a percentage of carbon compounds capable of complexing these metals (10-12). This paper reports the results of studies designed to test the longevity of chlorine inhibition to marine phytoplankton and the possibility of increased sensitivity to trace metals. Materials and Methods Algal Species and Culture. A number of unialgal cultures of phytoplankton were maintained in f/2 medium (13) at 20%0salinity. Algal species were chosen to reflect 0013-936X/84/0918-0383$01.50/0
the various types of natural species found in the mesohaline portion of estuaries and included a centric diatom, Thalassiosira pseudonana (clone 3H),a chrysophyte, Isochrysis galbana (TISO), and a green flagellate, Dunaliella sp. (DUN). Initial clones were obtained from the Culture Collection of Marine Phytoplankton of the Bigelow Laboratory for Ocean Sciences. Week-old stock cultures of these species were used as inocula in the series of bioassays described below. Chlorine Bioassays. Estuarine water from the mesohaline portion of the Patuxent River, a subestuary of Chesapeake Bay, was filtered through acid-washed glass fiber filters, autoclaved, and split into two aliquots. One aliquot was chlorinated (10 mgL-l) with NaOC1, resulting in a “control” and a “chlorinated” seawater. This water was then aged for 35 days at 20 “C in polypropylene carboys. Residual chlorine concentrations were measured by using amperometric techniques (14) immediately after chlorination to verify dosage and at the completion of the aging period. After this period, each water type was enriched with N, P, and Si to concentrations equivalent to that of f/10 and used as a growth medium. Separate portions of these media were inoculated with the three species of algae noted above. Bioassays were performed in 50-mL glass test tubes by using a modified technique of Murphy and Belastock (15). Algal inocula were added to the aged seawater media in concentrations sufficient to give an initial cell density of 1 X lo3cells-ml-l. Twenty-five milliliters of this culture was then placed into duplicate test tubes. In addition to these tubes, duplicate tubes were also prepared as above with additions of inorganic copper (Cu) at concentrations of 10-100 pgL-1. Tubes were placed upright in an algal incubator at a light intensity of 65 pEinsteins.m-2-s-1,at 20 “C, and with a 12-12 light-dark cycle. In vivo fluorescence was recorded daily in a Turner Designs fluorometer following agitation of the tubes. Fluorescence doublings per day were used as a measure of growth. Significant differences in growth rates between treatments were detected by using analysis of variance. The above bioassay was repeated with the exception that the seawater was chlorinated and aged only 23 days. The bioassays were performed with only Dunaliella sp., but the number of different copper concentrations was increased to 5. In addition to measurement of residual chlorine compounds, the concentrations of dissolved organic carbon (DOC) in the water samples were measured both before and after chlorination and aging. This estimates the quantity of carbon oxidized by chlorine degradation (12). The first bioassay was repeated a third time, with the exception that the seawater was split into four aliquots and three levels of chlorination (0.5, 1.0, and 5.0 mgL-’) were employed. The four aliquots were then aged for 10 days. A similar series of bioassays was performed by using these aliquots and Dunaliella sp.
Results and Discussion The first series of bioassays contrasted the growth of three algal species in control and aged, chlorinated (10 rngL-l) water. Total residual chlorine was undetectable after the aging process. The response of the algae varied widely. Both I. galbana and T. pseudonana would not
0 1984 Amerlcan Chemical Society
Environ. Sci. Technol., Vol. 18, No. 5, 1984 383
Table I. Growth of Three Algal Species in Chlorinated and Unchlorinated Control Media under Varying Cu Concentrationsa algal species
added Cu, @.L-'
0.10.50 u
p
No Cu
fluorescence doublings-day-' control chlorinated
(A) Water Aged 35 days after Chlorination T. pseudonana 0 1.55 0
I. galbana
10 50 100
1.40* 0.32*
0
0.92 0.46* 0.03*
10 50 100
Dunaliella sp.
0 10 50 100
O*
O* 1.21 1.23 1.25 1.18
0 0 0
0 0 0 0 1.43 1.35 1.08* 0.90*
(B) Water Aged 23 days after Chlorination Dunaliella sp. 0 1.31 0 5 10 25 50 100
1.34 1.34 1.39 1.35 1.29
0 0 0 0 0
"Growth measured 24-72 h (see text); 10 mgL-' chlorination, water then aged before bioassays. Growth rates significantly (p C 0.05) less than control growth are indicated (asterisk).
grow at all in the chlorinated water, even after 35 days of aging (Table I). Their response to Cu additions in control water was typical of sensitive species, with significant growth inhibition occurring at all Cu concentrations employed. Dunaliella s ~ .on , the other hand, was quite resistant to Cu in control media; growth was not significantly inhibited by Cu concentrations as high as 100 pgL-l (Table IA and Figure 1). When grown in chlorinated media, however, all cultures exhibited a 24-h growth lag. In addition, Cu concentrations of 10 pgL-l reduced growth rates slightly, and higher concentrations reduced growth significantly (Figure 1 and Table IA). The second series of bioassays utilized only the resistant alga, Dunaliella sp., and water that had aged 23 days. As previously stated, total residual chlorine was undetectable. The highest Cu concentration reduced cell growth slightly (2%), but the reduction was not statistically significant (Table IB). However, Dunaliella sp. could not grow at all in the chlorinated water. The chlorinated water contained less DOC, 2.31 f 0.07 vs. 2.70 f 0.19 mg.L-l, than the control. This loss of DOC, 0.39 mgL-l or 32 pmolmL-l, is comparable to average losses of 35 pmo1.L-l in a related study of the Patuxent River (12). The third series of bioassays was performed by using only Dunaliella sp., three chlorination levels (0.5, 1.0, and 5.0 mgL-l), and water that had aged 10 days. Total re: sidual chlorine was undetectable in all chlorinated waters after the 10-day period. As earlier, no significant reductions in growth due to Cu dosage were observed in control media. As the level of chlorination increased, the degree of growth reduction caused by Cu increased, with growth significantly reduced by 100 pgL-l at chlorination levels of 0.5 and 1.0 mgL-l and by 50 pgL-' at chlorination levels of 1.0 mg-L-l (Table 11). Chlorination to 5.0 mg-L-' resulted in complete growth inhibition. Because the degradation of chlorine-produced oxidants in seawater is very rapid (14,16,17),an incubation or aging period of 10 days was considered sufficient to remove re384
Environ. Sci. Technol., Vol. 18, No. 5, 1984
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Figure 1. Growth of Dunaliella sp. in control (-)
and chlorinated (---) water at various Cu concentrations. Initial cell densities were 1 X lo3 cells.mL-'. The vertical bars represent the range of replicate in vivo fluorescence measurements.
Table 11. Growth of Dunaliella sp. in Chlorinated and Unchlorinated Control Media under Varying Cu Concentrationsa added Cu, pgL-' 0 10 50 100
fluorescence doublings-day-' 0.50 1.0 5.0 control mgL-' mgL-' mgL-' 1.48 1.44 1.48 1.29
1.60 1.60 1.64 1.38*
1.44 1.50 1.31* 1.07*
0 0 0 0
"Growth measured 24-72 h (see text). Water aged 10 days after chlorination. Growth rates significantly (p C 0.05) less than control growth are indicated (asterisk).
sidual products from the seawater used for bioassays. In a large number of experiments performed with water of similar salinities, no residual products were ever detected after 10 days (12). However, the seawater chlorinated for use in the first series of bioassays was aged 35 days. Even after this period, two of the three algal species would not grow in the chlorinated media. The resistant alga, Dunaliella sp., grew as well in the chlorinated media as in controls, but its resistance to other stresses, e.g., Cu, was lowered. In addition, Dunaliella growth in chlorinated media followed a 24-h lag, while the cultures in nonchlorinated media had no lag period (Figure 1). In another bioassay series with water aged 23 days, even Dunaliella sp. would not grow in chlorinated water. The degree of chlorination, as well as the aging time, was important in determining cell growth. After 10 days of aging, Dunaliella sp. would not grow in water chlorinated at 5.0 mgL-l, but it grew as well in water chlorinated at 0.50-1.0 rng-L-l as in nonchlorinated controls. It was, however, more sensitive to Cu stress, and increasing the level of chlorination caused larger reductions in growth because of Cu dose (Table 11).
The increased sensitivity of Dunaliella sp. to Cu may be due to increased activity of Cu2+(the toxic Cu species) because of oxidation of carbon compounds capable of complexing metal ions (12). Copper complexation capacities ranging from 0.1 to 0.4 pmo1-L-l have been measured in the Patuxent River (18). The degradation of chlorine in natural waters is accompanied in most instances by a loss of carbon (12,19,20). Water from the Patuxent River averaged a 35 pmo1.L-l loss when chlorinated in a parallel study (12) and a 32 pmol-L-l loss during this study. Assuming a 1:l stoichiometry between Cu and carbon functional groups and that the functional groups responsible for Cu complexation are attacked during organic oxidation by chlorine, the degree of carbon oxidation caused by chlorination is sufficient to destroy all of this complexation capacity. Increased Cu2+activity may not be the sole mechanism of inhibition, however; persistent chlorination byproducts may also be present. There is obviously still an inhibitory effect of chlorination, even after 35 days of aging, because two species of algae failed to grow regardless of Cu dose. It is logical that one or a number of toxic products persist in chlorinated estuarine waters at levels below detection by conventional techniques. The identification and investigation of the prevalence and toxicity of these compounds warrant further study. The levels of chlorination studied above are comparable to concentrations employed by industries that utilize estuarine water for cooling purposes ( 1 mg.L-l) and by municipalities chlorinating sewage effluent before discharge into receiving waters (1-10 mgL-l). The longevity of inhibitory response noted in these laboratory experiments can be extrapolated to natural systems, therefore, underscoring the potential for alteration of plankton communities. In the Patuxent River, for example, a large portion of the river flow in the mesohaline section is chlorinated as it passes through the cooling system of the Chalk Point Power Station, especially in years of low flow (21). Thus, this chlorination response can be widespread within an estuary. In addition, earlier studies suggesting that chlorination can lead to alterations in community structure (5, 6) as well as reduced productivity are supported by the varied response to chlorinated water exhibited by the three algal species used in the present studies. Chlorination is not the only stress to which an estuarine phytoplankton community may be subjected. These results suggest that chlorination can increase a species’ sensitivity to an additional stress (in this case, a toxic metal), if both are present. Because estuaries generally will be subjected simultaneously to a number of stresses, this added sensitivity is likely to occur. N
Acknowledgments I thank S. J. Cibik for culture maintenance and for
performing many of the bioassays and K. G. Sellner and L. W. Hall, Jr., for comments and criticism.
Literature Cited (1) Hall, L. W., Jr.; Helz, G. R.; Burton, D. T. “Power Plant Chlorination-A Biological and Chemical Assessment”;Ann Arbor Science : Ann Arbor, MI, 1981; p 237. (2) Eppley, R. W.; Renger, E. H.; Williams, P. M. Estuarine Coastal Mar. Sei. 1976,4 , 147-162. (3) Goldman, J. C.; Davidson, J. A. Environ. Sci. Technol. 1977, 11, 908-913. (4) Briand, F. J. P. Mar. Biol. 1975,33, 135-146. (5) Videau, C.; Khalanski, M.; Penot, M. J . Exp. Mar. Biol. Ecol. 1979,36, 111-123. (6) Sanders, J. G.; Ryther, J. H. In “Water ChlorinationEnvironmental Impact and Health Effects”; Jolley, R. L.; Brungs, W. A,; Cumming, R. B., Eds.; Ann Arbor Science: Ann Arbor, MI, 1980; Vol. 3, pp 631-639. ( 7 ) Goldman, J. C.; Capuzzo, J. M.; Wong, G. T. F. In “Water Chlorination-Environmental Impact and Health Effects”; Jolley, R. L.; Gorchev, H.; Hamilton, D. H., Eds.; Ann Arbor Science: Ann Arbor, MI, 1978; Vol. 2, pp 291-305. (8) Goldman, J. C.; Quinby, H. L. J. Water Pollut. Control Fed. 1979,51,1816-1823. (9) Sanders, J. G.; Ryther, J. H.; Batchelder, J. H. J.Exp. Mar. Biol. Ecol. 1981,49,81-102. (10) Carpenter, J. H.; Smith, C. A. In “Water ChlorinationEnvironmental Impact and Health Effects”; Jolley, R. L.; Gorchev, H.; Hamilton, D. H., Eds.; Ann Arbor Science: Ann Arbor, MI, 1978; pp 195-207. (11) Carpenter, J. H.; Smith, C. A.; Zika, R. J. “Reaction Products from the Chlorination of Seawater”. U. S. Environmental Protection Agency, Washington, DC, 1981, Final Report EPA-600/S4-81-010. (12) Sanders, J. G. Environ. Sci. Technol. 1982,16,791-796. (13) Guillard, R. R. L.; Ryther, J. H. Can. J . Microbiol. 1962, 8, 229-239. (14) Goldman, J. C.; Quinby, H. L.; Capuzzo, J. M. Water Res. 1979,13,315-323. (15) Murphy, L. S.; Belastock, R. A. Limnol. Oceanogr. 1980, 25, 160-165. (16) Wong, G. T. F.; Davidson, J. A. Water Res. 1977, 11, 971-978. (17) Wong, G. T. F. Water Res. 1982,16,335-343. (18) Newell, A. D. M.S. Thesis, University of North Carolina, Chapel Hill, NC, 1983, p 97. (19) Sigleo, A. C.; Helz, G. R.; Zoller, W. H. Environ. Sei. Technol. 1980,14,673-679. (20) Helz, G. R.; Dotson, D. A,; Sigleo, A. C. In “Water Chlorination-Environmental Impact and Health Effects”; Jolley, R. L; Gorchev, H.; Hamilton, D. H., Eds.; Ann Arbor Science: Ann Arbor, MI, 1983; Vol. 4, pp 181-189. (21) Eaton, A.; Chamberlain, C. “Cu Cycling in the Patuxent Estuary”. Department of Natural Resources, Power Plant Siting Program, MD, 1980, Final Report P42-78-04.
Received for review September 20, 1983. Accepted December 21,1983. This research was supported by the Maryland Department of Natural Resources, Power Plant Siting Program (P708004),and the Academy of Natural Sciences.
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