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Comparison of radiocarbon uptake and DCMU-fluorescence techniques in evaluating dispersed oil effects on phytoplankton photosynthetic activity
Wat. Res. Vol. 25, No. 10, pp. 1249-1254, 1991 Printed in Great Britain.All fights reserved
0043-1354/91 $3.00+ 0.00 Copyright © 1991 Pm'garnonPrtmspie
COMPARISON OF RADIOCARBON UPTAKE A N D DCMU-FLUORESCENCE TECHNIQUES IN EVALUATING DISPERSED OIL EFFECTS ON PHYTOPLANKTON PHOTOSYNTHETIC ACTIVITY SUZANNE ROY, ROBERT SIRON and ]~MILIENPELLETIER~ Centre O~anographique de Rimouski, INRS-Oc~nologie, 310 AII~¢ des Ursulines, Rimouski, Quebec, Canada G5L 3A1 (First received August 1990; accepted in revised form March 1991)
Al~ract--In a mesocosm study of dispersed-oil effects on cold seawater natural phytoplankton, two methods were compared for assessing algal photosynthesis. The radiocarbon (14C)uptake method seemed to be affected by elevated heterotrophie activity caused by a bacterial increase a few days after oil addition and by the presence of small dispersed oil droplets retained on the filters. These artefacts are thought to be the cause of an apparent stimulation of the specific photosynthetic rate (P/B, or photosynthetic rate per unit algal biomass) following oil contamination, although actual photosynthetic rates declined in all cases after contamination. Such artefacts did not affect the DCMU-fluorescence response, showing a decrease in photosynthetic ability that followed the decline in chlorophyll a concentration and was very sensitive, whatever the initial algal biomass. The latter technique thus seems more appropriate for assessment of deleterious effects on phytoplankton photosynthesis, especially in oligotrophie environments. Addition of dispersed crude oil at concentrations of the order of 10 nag 1-l in April and 4 nag 1-i in December resulted in a decline in phytoplankton biomass and photosynthetic ability. Progressive dilution of the contaminated environment helped recovery from toxic effects after 1 week. Key words---dispersed oil, marine phytoplankton, photosynthetic activity, 14C uptake, DCMUfluorescence, mesocosms, cold seawater
INTRODUCTION
As part of an exhaustive study on the effects of dispersed crude oil in cold marine waters, an investigation of the response of natural phytoplankton communities enclosed in mesocosms was carried out. As the presence of dispersed oil droplets in seawater had an unknown effect on the techniques generally used to evaluate this response (Cross, 1982), results from different techniques were compared, including chlorophyll a concentration and microscopic cell counts for phytoplankton biomass, and in situ radiocarbon (14C) uptake and DCMU*-enhanced fluorescence measurements for photosynthetic activity. Radiocarbon uptake (Steemann Nielsen, 1952; Parsons et al., 1984) is a well known method in pollution-related work but the DCMU-fluorescence technique (Samuelsson and Oquist, 1977) has received less attention. In this technique, a metabolic inhibitor of electron transport in photosynthesis (DCMU) is added and/n vivo fluorescence is measured before and after this addition. DCMU-inhibition causes an increase in fluorescence which is roughly proportional to the fluorescence that was quenched by photochemistry prior to addition of the inhibitor and can be taken as a measure of actual *DCMU = 3-(3,4-dichlorophenyl)- 1,1-dimethylurea.
photosynthetic capacity (Samuelsson and Oquist, 1977, Roy and Legendre, 1979). Although strict biophysical interpretation of this technique is sometimes difficult (Falkowski and Kiefer, 1985), it is considered a reliable indicator of photosynthetic capacity (Cullen et aL, 1986) and is technically rapid and easy to use. The work presented here focuses on a comparison between the two methodologies used for measuring phytoplankton photosynthetic activity and an assessment of their usefulness in seawater contaminated with dispersed oil. Additionally, it provides information concerning the response of natural phytoplankton assemblages to additions of various concentrations of dispersed Forties crude oil under field conditions in winter and early spring in enclosed experimental ecosystems (mesocosms). Previous work on seawater contamination by crude oil has shown that effects of oil on phytoplankton can be both stimulatory and inhibitory (Vandermeulen and Ahem, 1976), depending on the concentration and the nature of the oil, and that the use of chemical dispersants often aggravates toxic effects on phytoplankton (Hsiao et al., 1978; Scbolten and Kuiper, 1987). The aim of this part of the study was thus to determine the effects of crude oil dispersion on winter (December) and early spring (April) natural phytoplankton taken from the St Lawrence estuary (Qutbec, Canada).
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Fig. 1. Cascade mode operation set-up, as used in the April experiment: dispersed crude oil was added at a tank depth of 2 m in tank C-1 and peristaltic pumps were used to transfer daily 16% of the tank's volume between tanks C-I and C-2 and between tanks C-2 and C-3. The volume of tank C-1 was maintained by re-supplying it from an uncontaminated seawater supply. MATERIALS AND METHODS Mesocosms
In both experiments described below, five mesocosms of 3.5 m 3 each were filled with unfiltered seawater originating from a depth of 3 m, collected directly off the coast at Pointe-au-P~re (Qu6bec, Canada) on 12 April 1989 for the first experiment, which lasted 17 days, and on 30 November 1989 for the second one, of 13 days duration. These mesocosms are 3 m-tall double-walled cylinders with an inner diameter of 1.4 m and they are temperature-controlled using a recirculating coolant (Petletier et al., 1989). The top part of the cyclinders is exposed to natural outdoor light, covered only with a Plexiglas sheet to protect them from rain and snow. Seawater temperature in the tanks varied between 2 and 5°C in the April experiment and from - 2 to - I ° C in
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the December one. For about 1 week before introduction of the oil~lispersant mixture, seawater was circulated between the five tanks using peristaltic pumps, to ensure homogeneity and allow natural phytoplankton growth to take place. Nutrients (nitrate, phosphate and silicate salts) were monitored regularly using a Teehnicon Auto-Analyzer system (Strickland and Parsons, 1972). When necessary, these nutrient salts were added to prevent nutrient depletion when phytoplankton growth was strong. Mixing was maintained in each tank by gentle air bubbling. Two modes of operation were used, "static" in April, "cascade" in December. In "static" mode, water circulation between the tanks was stopped before introduction of the contaminant, isolating the five mesocosms. Three tanks were then treated with different concentrations of a pre-mixed oil-dispersant solution. The oil~lispersant mixture was introduced into the tanks at a depth of 2 m. In "cascade" mode, only one tank was treated, but water circulation was continued afterwards between this first tank and two others, placed in series, thus progressively contaminating these other two tanks (Fig. l). The turnover volume was 16% of the tank's volume. To avoid volume depletion, the first tank was replenished with uncontaminated nonfiltered seawater pumped from the same site as the original water used to fill the tanks. This cascade set-up was used as a model for simulation of oil spills, with gradual contamination of neighboring sites and dilution of the spill area. In the April experiment, pre-mixed oil--dispersant solution (10:1 Forties crude oil and Corexit 9527) was introduced at an initial concentration of approx. 100 ppm for tank I, 20 ppm for tank 2 and 5 ppm for tank 3. Measured total oil concentrations in seawater can be found in Fig. 2. The remaining two tanks received no oil or dispersant and were used as controls. In the December experiment, pre-mixed oil-dispersant mixture was composed of Forties crude oil and a combination of three dispersing agents (AOT, Brij 96 and Brij 92) in the ratio 10:1. This was introduced in the first tank of the cascade, at a nominal concentration of 150 ppm (measured oil concentrations: Fig. 2). Analytical methods
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Every 2 or 3 days, the following variables were measured: (1) in vivo and DCMU-fluorescence (Roy and Legendre, 1979), determined on a Turner Designs 10-005 R fluorometer, after 15 min dark adaptation (DCMU final concentration = 3/~M); (2) chlorophyll a, extracted with 90% acetone from Whatman GF/F filter-collected material and analyzed fiuorometrically according to Yentseh and Menzel (1963); (3) 14C uptake, following the method described in Parsons et aL (1984), using 0.1 ml of a I0 #Ci (370 klkl) ml -t NaH~4CO3 solution added to 25 ml glass vials (April experiment) or 300 ml bottles (December experiment: size of bottles adjusted according to phytoplankton concentration in the tanks) filled
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Fig. 3. Daily changes in chlorophyll a concentration (~g.l -l) in the April and December experiments. Labels for individual tanks are explained in the text. Day 0 refers to oil addition. DO = dispersed oil, C = cascade. with seawater from 1 m depth in the tanks. In situ incubation of two light and one dark bottle lasted 2-4 h, filtration was done on Millipore AAWP filters (porosity = 0.8 #m) which were rinsed with filtered seawater, put in vials and acidified with I ml of 0.1 N HCI. Aquasol (12 ml) was added 2 h later and counting was done with a scintillationcounter (Beckman LS 5801). Frequency of sampling for 14Cuptake was once or twice weeklydue to the time requirements for this technique; (4) lugol-preserved phytoplankton samples, for taxonomic identification; (5) formalin-preserved water samples, for bacterial counts using the epifluorescence acridine orange technique (Hobbie et al., 1977), and (6) total (particulate + dissolved) oil concentration in seawater, by u.v. absorption at 233 nm of dichloromethane extracts. RESULTS
Chlorophyll measurements and taxonomic observations
In the April experiment, strong growth of phytoplankton took place in the five tanks before addition of the dispersed oil on day 0 (Fig. 3). Circulating seawater between the tanks before the beginning of the experiment resulted in relative inter-tank homogeneity in chlorophyll a concentration (cf. day - 1, Fig. 3). After addition of dispersed oil in the three experimental tanks, divergence in the chlorophyll a concentration was soon observed: the DO-100 ( = dispersed oil - 100 ppm) tank showed an immediate leveling off of the chlorophyll a concentration. The decrease in total oil concentration in that tank, down to about 10 ppm after 12 days (Fig. 2), had no effect on the chlorophyll a concentration which continually declined until the end of the experiment. This decline was accompanied by a loss in all previously WR
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Fig. 4. Daily changes in the specific photosynthetic rate (P/B), chlorophyll a concentration (CHL) and DCMUfluorescence increase (Fd-F.), expressed as percent value of controls for the April experiment. Values above 100% (i.e. identical to controls) indicate a stimulation; those below 100% show an inhibition. dominant phytoplankton species (mostly diatoms). The DO-5 (--5 ppm) tank is the only contaminated tank where chlorophyll = concentrations generally remained between those of the two control tanks. Controls diverged in terms of chlorophyll a concentration 4 days after the beginning of the experiment. This may be related to a change in composition of the dominant algal populations after day 4. Diatoms gave way to small unidentified flagellates ( < 10#m) after day 4 in the CTRL-2 ( = control No. 2) tank. Since we had no reason to consider results from either control tanks unreliable, control values were calculated from the mean of results from the 2 control tanks. The 3 experimental tanks were then compared to these control values and a percent control was calculated as the ratio between values obtained from each experimental tank and the means of the 2 control tanks (Fig. 4). In the December experiment, chlorophyll a concentrations were much lower than in the April experiment (Fig. 3). Before the start of the experiment, concentrations varied between 0.5 and 0.8 gg chl a . l-1. This was associated with a very low cell concentration of phytoplankton, the dominant algal populations being micro-flagellates. During the first week after introduction of the dispersed oil mxture, the tank initially contaminated (tank C-h head of cascade) and the
SUZANNE ROY et al.
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Radiocarbon uptake
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Fig. 5. Daily changes in the specific photosynthetic rate (P/B), chlorophyll a concentration (CHL) and D C M U fluorescence increase ( F d - F n ) , expressed as percent value of controls for the December experiment. s e c o n d t a n k o f the c a s c a d e (tank C-2) s h o w e d generally lower c h l o r o p h y l l a c o n c e n t r a t i o n s ( a r o u n d 0 . 2 g g . l - ] ) t h a n the o t h e r t a n k s (Figs 3 a n d 5). H o w e v e r , results f r o m c o n t r o l a n d e x p e r i m e n t a l t a n k s were n o t as clearly s e p a r a t e d as in the April experiment.
In the April e x p e r i m e n t , specific p h o t o s y n t h e t i c rate ( = c a r b o n u p t a k e p e r unit c h l o r o p h y l l a ) gave values a b o v e control, especially for the m o s t c o n t a m i n ated t a n k 1 week after c o n t a m i n a t i o n (Fig. 4 a n d Table 1). S t i m u l a t i o n o f the p h o t o s y n t h e t i c activity c o n t r a s t s with t h e previously d e s c r i b e d i n h i b i t i o n o f c h l o r o p h y l l b i o m a s s (Fig. 3). A c t u a l r a d i o c a r b o n uptake values were still 16.6 m g C m -3 h -], 8 days after c o n t a m i n a t i o n , while chlorophyll a c o n c e n t r a t i o n h a d declined f r o m 37.3 o n d a y 0 to 8.8 m g m -3 (Table 1). A s in the A p r i l e x p e r i m e n t , specific p h o t o s y n t h e t i c rates o f the D e c e m b e r e x p e r i m e n t also s h o w e d values a b o v e control, especially for t a n k s C-1 a n d C-2, the t w o m o s t c o n t a m i n a t e d t a n k s (Fig. 5). This stimulation o f the p h o t o s y n t h e t i c activity again was n o t m a t c h e d by the c h l o r o p h y l l d a t a (Fig. 3).
DCMU-fluorescence In the A p r i l e x p e r i m e n t , D C M U - f l u o r e s c e n c e s h o w e d a d e c r e a s e in p h o t o s y n t h e t i c ability relative to c o n t r o l s in t a n k s D O - 1 0 0 a n d D O - 2 0 following c o n t a m i n a t i o n (Fig. 4). D C M U - f l u o r e s c e n c e generally f o l l o w e d the s a m e t r e n d as the c h l o r o p h y l l a c o n c e n tration. In the D O - 1 0 0 t a n k , D C M U - f l u o r e s c e n c e generally r e m a i n e d b e l o w 2 0 % o f c o n t r o l values, while at the s a m e time, a c c o r d i n g to the r a d i o c a r b o n u p t a k e m e t h o d , p h y t o p l a n k t o n p h o t o s y n t h e s i s was stimulated. In t h e D e c e m b e r e x p e r i m e n t , D C M U - f l u o r e s c e n c e s h o w e d a decrease relative to c o n t r o l s in the cont a m i n a t e d t a n k s C-1 a n d C-2 (Fig. 5) a n d this t r e n d generally f o l l o w e d t h a t o f the c h l o r o p h y l l a c o n c e n tration. In the initially c o n t a m i n a t e d t a n k ( C - l ) , D C M U - f l u o r e s c e n c e values d e c r e a s e d to zero 1 d a y after i n t r o d u c t i o n o f t h e o i l - d i s p e r s a n t m i x t u r e a n d r e m a i n e d at this value until d a y 8. In c o n t r a s t , r a d i o -
Table I. Primary production data. Only the most contaminated tank and control tanks are shown April experiment: Days after contamination Tank PP-light PP-dark D/L B P/B 2 DO-100 85.42 __.3.98 37.40 44% 31.80 + 3.18 2.69 + 0.40 CTRL 216.84 _ 24.66 39.91 + 0.91 18% 88.42 + 2.14 2.45 _+0.34 8 DO-100 16.62 + 0.45 2.31 14% 8.77 ___0.88 1.90 + 0.24 CTRL 57.47+13.12 0.76+0.40 1% 110.78_+91.25 0.71_+0.47 15 DO-100 1.08 _+0.02 0.53 49% 1.09 + 0.11 0.99 -+ 0.12 CTRL 60.66 + 62.11 0.49 -+ 0.57 1% 44.25 ___55.2.8 2.40 _+ 1.62 December experiment: Days after contamination Tank PP-light PP-dark D/L B P/B 1 C-I 0.21 + 0.08 0.20 95% 0.11 1.90 + 0.74 CTRL 0.15 + 0.10 0.13 87% 0.34 0.24 + 0.27 4 C-I 0.65 -+_0.51 0.70 108% 0.09 7.22 -+ 5.65 CTRL 0.19 + 0.10 0 0% 0.23 0.83 + 0.44 6 C-1 0.04 + 0.002 0 0% 0 0 CTRL 0.05 + 0.004 0 0% 0.12 + 0.16 0.42 + 0.63 8 C-I 0.10 + 0.02 0.09 87% 0.11 + 0.07 0.88 + 0.73 CTRL 0.02 + 0.003 0 0% 0.09 + 0.10 0.25 + 0.31 11 C-I 0.09 + 0.03 0.19 210% 0.20 _+0.05 0.45 + 0.26 CTRL 0.06 + 0.03 0 0% 0.10 + 0.01 0.61 _ 0.37 PP-light ffiprimary production (mg C. m3.h- ~) in the light bottles; PP-dark = results from the dark bottles; D/L = percent ratio of PP-light/PP-dark; B = chlorophyll a concentration (mg.m-~); P/B = specific photosynthetic rate (rag C. mg Chl- '. h -' ); value for the controls (CRTL) is the average of the P/B of the 2 control tanks. When replicate measurements were made (as in the 2 light bottles for PP), average values are indicated _+SD.
DCMU-fluorescence and l'C-uptake in oiled reservoirs carbon uptake showed a stimulation of photosynthetic activity relative to controls during this period. DISCUSSION
Techniques comparison The two techniques used here to evaluate phytoplankton photosynthetic activity do not, in fact, measure the same thing: the radiocarbon uptake method gives a value of the rate of carbon uptake on a time scale of hours, while the DCMU-fluorescence method gives information on the pool size of active photosystem II reaction centers (Falkowski and Kiefer, 1985). DCMU-fluorescence reflects the photosynthetic ability of the cell (Vincent, 1980) by expressing the potential for photochemistry and it can be useful as a fast and simple method to indicate inhibition of photosynthesis in cases of contamination (Cullen et al., 1986). In this work, it is surprising that photosynthetic ability (DCMU-fluorescence) seems to be inhibited while photosynthetic rate is stimulated, particularly considering the high concentration of oil used. Closer examination of the data (Table 1) shows that, while both photosynthetic rate (P) and chlorophyll concentration (B) are generally reduced after the oil treatment, the specific photosynthetic rate ( P / B ) is often higher than controls because the reduction in P is generally less than that in B. Highe r than expected 14C uptake values could have resulted in the oiled samples if heterotrophic activity was strong. This is in fact observed here: uptake measured in dark-incubated bottles (Parsons et al., 1984) is quite high (Table 1) and this has been noted in other oil studies (Cross, 1982; Dahl et al., 1983). A bacterial increase following oil contamination likely contributed to this heterotrophic uptake, particularly in the April experiment (Fig. 6). In addition, dispersed oil droplets of 2080 #m size (Coulter particle size spectra, data not presented) were also observed in significant concentralOOO- '
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tion in the oiled tanks and may have contributed to non-algal uptake of the radioactive tracer by physical adsorption on the droplets and retention on the 0.8 #m filters (not actually verified). These effects may be particularly important when the concentration of photosynthetic material is low, as in the December experiment. DCMU-fluorescence on the other hand, was not affected by either the presence of bacteria or dispersed oil particles or by the low concentration of algae present. Replicability of measurements was better (coefficient of variation less than 10%) than with the radiocarbon uptake method (Table 1) even in highly oligotrophic conditions. The dispersed oil fluoresces at a low level under the conditions used for measuring chlorophyll fluorescence in algae (blanks below 0.5 relative fluorescence units) and measurement of the increase in fluorescence after addition of DCMU effectively removes any background effects. This technique has been used successfully in a few other studies dealing with oil contamination (Ostgaard et al., 1984; Siron et al., 1991), with copper toxicity (Cullen et al., 1986) and in mesocosm studies (Keller, 1987). In comparing the two methods used in this study to assess dispersed oil effects on phytoplankton photosynthesis, it appears that the DCMU-fluorescence method is more appropriate and convenient than the radiocarbon uptake method on the following grounds: (1) it is less time-consuming, (2) it does not require the use of radioactive substances, (3) the equipment needed is not expensive and is easy to use in the field, (4) it is not affected by heterotrophic activity or the presence of oil particles, and (5) replicability of measurements is high. Effects of dispersed oil addition on phytoplankton In the April experiment, comparable to a spring bloom condition, dispersed oil introduced in seawater at a concentration close to 10mg.l -~ caused a net decline in chlorophyll a and cell biomass, as well as in the photosynthetic ability of natural phytoplankton enclosed in mesocosms. In the static mode used in this experiment, the effects of contamination lasted as long as the experiment, i.e. there were no obvious signs of recovery. In the December experiment, the actual concentration of total oil in seawater was much lower: this was probably related to a decreased efficiency of the dispersant used in very cold waters and to rapid sedimentation of the added oil before dispersion could act (Siron et al., in preparation). Nonetheless, with oil concentrations around 4 mg. 1-i (tank C-1), a decrease in chlorophyll a biomass and in photosynthetic ability was observed. Although the decrease in chlorophyll biomass was not very distinct from the variations found in the other tanks, probably due to the very low values which are normal for this time of the year in the natural environment (Therriault and Levasseur, 1985), the inhibition of photosynthetic ability in tank C-1 was very clear (Fig. 5). In this December experiment, where mesocosms were run in the cascade
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SUZANNEROY et al.
mode, some signs of recuperation from contamination were detectable after 1 week in the most contaminated tank (Fig. 5), suggesting that residual oil concentrations were not lethal after that time and that dilution can contribute to alleviate toxic effects from dispersed oil. These responses to oil addition are consistent with findings from previous studies run under comparable conditions, i.e. with natural phytoplankton and under field conditions. Concentrations of crude oil-seawater dispersion of the order of 10mg.1-1 and less have been shown to cause deleterious effects on biomass and photosynthetic activity of natural phytoplankton, both in non-renewed large enclosures (Harrison et al., 1986) and in flow-through systems (Linden et al., 1985). As already shown for arctic marine phytoplankton (Hsiao et al., 1978), the work presented here indicates that natural assemblages of phytoplankton from north-temperate environments, such as the St Lawrence estuary in mid-winter or early spring periods, can suffer from contamination with chemically-dispersed oil at relatively low concentrations and that both biomass and photosynthetic activity can be affected. Acknowledgements--The authors wish to thank C. Brochu, L. Bernier, N. Blouin and F. Btlanger for technical assistance. This research was funded by the National Sciences and Engineering Research Council of Canada through its strategic grant program (grant No. STRGP 036). This publication is a contribution of the Oceanographic Centre of Rimouski--a partnership of INRS (Institut National de la Recherche Scientifique) and UQAR (Universit6 du Quebec ~ Rimouski) operating under the auspices of the University of Qutbec.
REFERENCES
Cross W. E. (1982) In situ studies of effects of oil and dispersed oil on primary productivity of ice algae and on under-ice amphipod communities. Special studies--1981 study results, (BIOS) Baffin Island Oil Spill Working Report 81-10. Cullen J. J., Zhu M. and Pierson D. C. (1986) A technique to assess the harmful effects of sampling and containment for determination of primary production. Limnol. Oceanogr. 31, 1364-1373. Dahl E., Laake M., Tjessem K., Eberlein K. and Bahle B. (1983) Effects of Ekofisk crude oil on an enclosed planktonic ecosystem. Mar. Ecol. Prog. Ser. 14, 8t-91. Falkowski P. G. and Kiefer D. A. (1985) Chlorophyll a fluorescence in pbytoplankton: relationship to photosynthesis and biomass. J. Plankton Res. 7, 715-731. Harrison P. J., Cochlan W. P., Acreman J. C., Parsons T. R., Thompson P. A. and Dovey H. M. (1986) The effects of crude oil and Corexit 9527 on marine phytoplankton in an experimental enclosure. Mar. envir. Res. 18, 93-109.
Hobble J. E., Daley R. J. and Jasper S. (1977) Use of Nuclepore filters for counting bacteria by fluoresctmee microscopy. Appl. envir. Microbiol. 33, 1225-1228. Hsiao S. I. C., Kittle D. W. and Foy M. G. 0978) Effects of crude oils and the oil dispersant Corexit on primary production of Arctic marine phytoplankton and seaweed. Envir. Pollut. 15, 209-221. Keller A. A. (1987) Mesocosm studies of DCMU-enhanced fluorescence as a measure of phytoplankton photosynthesis. Mar. Biol. 96, 107-114. Linden O., Rosemarin A., Lindskog A., Hogiund C. and Johansson S. (1985) Ecological effects of oil versus oil plus dispersant on the littoral ecosystem of the Baltic Sea. In Proceedings of the 1985 Oil Spill Conference, pp. 485490. American Petroleum Institute Publication No. 4385, Washington, D.C. Ostgaard K., Eide I. and Jensen A. (1984) Exposure of phytoplankton to Ekofisk crude oil. Mar. envir. Res. 11, 183-200. Parsons T. R., Maita Y. and Lalli C. M. (1984) A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press, Oxford. Pelletier 1~., Brochu C., Roy S. and Mayzaud P. (1989) New protected experimental tanks for environmental studies under severe weather conditions. Proceedings of the 1989 Oil Spill Conference, San Antonio, Tex. Roy S. and Legendre L. (1979) DCMU-enhanced fluorescence as an index of photosynthetic activity of phytoplankton. Mar. Biol. 55, 93-101. Samuelsson G. and Oquist G. (I977) A method for studying photosynthetic capacities of unicellular algae based on in vivo chlorophyll fluorescence. Physiol. PI. 40, 315-319. Scholten M. and Kuiper J. (1987) The effects of oil and chemically dispersed oil on natural phytoplankton communities. Proceedings of the 1987 Oil Spill Conference, pp. 255-257. Baltimore, Md. Siron R., Giusti G., Berland B., Morales-Loo M. del R. and Pelletier ]~. (1991) Water soluble petroleum compounds: chemical aspects and effects on the growth of microalgae. ScL Total Envir. In press. Steemann Nielsen E. (1952) The use of radioactive carbon (14C) for measuring organic production in the sea. J. Cons. perm. int. expl. Mer. l& 117-140. Strickland J. D. H. and Parsons T. R. (1972) A practical handbook of seawater analysis. Fish. Res. Bd Can. Bull. 167, t-310. Therriault J.-C. and Levasseur M. (1985) Control of phytoplankton production in the lower SL Lawrence estuary: light and freshwater runoff. Natn. Can. (Rev. Ecol. Syst.) 112, 77-96. Vandermeulen J. H. and Ahem T. P. (1976) Effect of petroleum hydrocarbons on algal physiology: review and progress report. In Effects of Pollutants on Aquatic Organisms (Edited by Lockwood A. P. M.), pp. 107-125. Cambridge University Press, Cambridge. Vincent W. F. (1980) Mechanisms of rapid photosynthetic adaptation in natural phytoplankton communities. II. Changes in photochemical capacity as measured by DCMU-induced chlorophyll fluorescence. J. Phycol. 16, 568-577. Yentsch C. S. and Menzel D. W. (1963) A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-Sea Res. 10, 221-231.
0043-1354/91 $3.00+ 0.00 Copyright © 1991 Pm'garnonPrtmspie
COMPARISON OF RADIOCARBON UPTAKE A N D DCMU-FLUORESCENCE TECHNIQUES IN EVALUATING DISPERSED OIL EFFECTS ON PHYTOPLANKTON PHOTOSYNTHETIC ACTIVITY SUZANNE ROY, ROBERT SIRON and ]~MILIENPELLETIER~ Centre O~anographique de Rimouski, INRS-Oc~nologie, 310 AII~¢ des Ursulines, Rimouski, Quebec, Canada G5L 3A1 (First received August 1990; accepted in revised form March 1991)
Al~ract--In a mesocosm study of dispersed-oil effects on cold seawater natural phytoplankton, two methods were compared for assessing algal photosynthesis. The radiocarbon (14C)uptake method seemed to be affected by elevated heterotrophie activity caused by a bacterial increase a few days after oil addition and by the presence of small dispersed oil droplets retained on the filters. These artefacts are thought to be the cause of an apparent stimulation of the specific photosynthetic rate (P/B, or photosynthetic rate per unit algal biomass) following oil contamination, although actual photosynthetic rates declined in all cases after contamination. Such artefacts did not affect the DCMU-fluorescence response, showing a decrease in photosynthetic ability that followed the decline in chlorophyll a concentration and was very sensitive, whatever the initial algal biomass. The latter technique thus seems more appropriate for assessment of deleterious effects on phytoplankton photosynthesis, especially in oligotrophie environments. Addition of dispersed crude oil at concentrations of the order of 10 nag 1-l in April and 4 nag 1-i in December resulted in a decline in phytoplankton biomass and photosynthetic ability. Progressive dilution of the contaminated environment helped recovery from toxic effects after 1 week. Key words---dispersed oil, marine phytoplankton, photosynthetic activity, 14C uptake, DCMUfluorescence, mesocosms, cold seawater
INTRODUCTION
As part of an exhaustive study on the effects of dispersed crude oil in cold marine waters, an investigation of the response of natural phytoplankton communities enclosed in mesocosms was carried out. As the presence of dispersed oil droplets in seawater had an unknown effect on the techniques generally used to evaluate this response (Cross, 1982), results from different techniques were compared, including chlorophyll a concentration and microscopic cell counts for phytoplankton biomass, and in situ radiocarbon (14C) uptake and DCMU*-enhanced fluorescence measurements for photosynthetic activity. Radiocarbon uptake (Steemann Nielsen, 1952; Parsons et al., 1984) is a well known method in pollution-related work but the DCMU-fluorescence technique (Samuelsson and Oquist, 1977) has received less attention. In this technique, a metabolic inhibitor of electron transport in photosynthesis (DCMU) is added and/n vivo fluorescence is measured before and after this addition. DCMU-inhibition causes an increase in fluorescence which is roughly proportional to the fluorescence that was quenched by photochemistry prior to addition of the inhibitor and can be taken as a measure of actual *DCMU = 3-(3,4-dichlorophenyl)- 1,1-dimethylurea.
photosynthetic capacity (Samuelsson and Oquist, 1977, Roy and Legendre, 1979). Although strict biophysical interpretation of this technique is sometimes difficult (Falkowski and Kiefer, 1985), it is considered a reliable indicator of photosynthetic capacity (Cullen et aL, 1986) and is technically rapid and easy to use. The work presented here focuses on a comparison between the two methodologies used for measuring phytoplankton photosynthetic activity and an assessment of their usefulness in seawater contaminated with dispersed oil. Additionally, it provides information concerning the response of natural phytoplankton assemblages to additions of various concentrations of dispersed Forties crude oil under field conditions in winter and early spring in enclosed experimental ecosystems (mesocosms). Previous work on seawater contamination by crude oil has shown that effects of oil on phytoplankton can be both stimulatory and inhibitory (Vandermeulen and Ahem, 1976), depending on the concentration and the nature of the oil, and that the use of chemical dispersants often aggravates toxic effects on phytoplankton (Hsiao et al., 1978; Scbolten and Kuiper, 1987). The aim of this part of the study was thus to determine the effects of crude oil dispersion on winter (December) and early spring (April) natural phytoplankton taken from the St Lawrence estuary (Qutbec, Canada).
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Fig. 1. Cascade mode operation set-up, as used in the April experiment: dispersed crude oil was added at a tank depth of 2 m in tank C-1 and peristaltic pumps were used to transfer daily 16% of the tank's volume between tanks C-I and C-2 and between tanks C-2 and C-3. The volume of tank C-1 was maintained by re-supplying it from an uncontaminated seawater supply. MATERIALS AND METHODS Mesocosms
In both experiments described below, five mesocosms of 3.5 m 3 each were filled with unfiltered seawater originating from a depth of 3 m, collected directly off the coast at Pointe-au-P~re (Qu6bec, Canada) on 12 April 1989 for the first experiment, which lasted 17 days, and on 30 November 1989 for the second one, of 13 days duration. These mesocosms are 3 m-tall double-walled cylinders with an inner diameter of 1.4 m and they are temperature-controlled using a recirculating coolant (Petletier et al., 1989). The top part of the cyclinders is exposed to natural outdoor light, covered only with a Plexiglas sheet to protect them from rain and snow. Seawater temperature in the tanks varied between 2 and 5°C in the April experiment and from - 2 to - I ° C in
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the December one. For about 1 week before introduction of the oil~lispersant mixture, seawater was circulated between the five tanks using peristaltic pumps, to ensure homogeneity and allow natural phytoplankton growth to take place. Nutrients (nitrate, phosphate and silicate salts) were monitored regularly using a Teehnicon Auto-Analyzer system (Strickland and Parsons, 1972). When necessary, these nutrient salts were added to prevent nutrient depletion when phytoplankton growth was strong. Mixing was maintained in each tank by gentle air bubbling. Two modes of operation were used, "static" in April, "cascade" in December. In "static" mode, water circulation between the tanks was stopped before introduction of the contaminant, isolating the five mesocosms. Three tanks were then treated with different concentrations of a pre-mixed oil-dispersant solution. The oil~lispersant mixture was introduced into the tanks at a depth of 2 m. In "cascade" mode, only one tank was treated, but water circulation was continued afterwards between this first tank and two others, placed in series, thus progressively contaminating these other two tanks (Fig. l). The turnover volume was 16% of the tank's volume. To avoid volume depletion, the first tank was replenished with uncontaminated nonfiltered seawater pumped from the same site as the original water used to fill the tanks. This cascade set-up was used as a model for simulation of oil spills, with gradual contamination of neighboring sites and dilution of the spill area. In the April experiment, pre-mixed oil--dispersant solution (10:1 Forties crude oil and Corexit 9527) was introduced at an initial concentration of approx. 100 ppm for tank I, 20 ppm for tank 2 and 5 ppm for tank 3. Measured total oil concentrations in seawater can be found in Fig. 2. The remaining two tanks received no oil or dispersant and were used as controls. In the December experiment, pre-mixed oil-dispersant mixture was composed of Forties crude oil and a combination of three dispersing agents (AOT, Brij 96 and Brij 92) in the ratio 10:1. This was introduced in the first tank of the cascade, at a nominal concentration of 150 ppm (measured oil concentrations: Fig. 2). Analytical methods
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Every 2 or 3 days, the following variables were measured: (1) in vivo and DCMU-fluorescence (Roy and Legendre, 1979), determined on a Turner Designs 10-005 R fluorometer, after 15 min dark adaptation (DCMU final concentration = 3/~M); (2) chlorophyll a, extracted with 90% acetone from Whatman GF/F filter-collected material and analyzed fiuorometrically according to Yentseh and Menzel (1963); (3) 14C uptake, following the method described in Parsons et aL (1984), using 0.1 ml of a I0 #Ci (370 klkl) ml -t NaH~4CO3 solution added to 25 ml glass vials (April experiment) or 300 ml bottles (December experiment: size of bottles adjusted according to phytoplankton concentration in the tanks) filled
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Fig. 3. Daily changes in chlorophyll a concentration (~g.l -l) in the April and December experiments. Labels for individual tanks are explained in the text. Day 0 refers to oil addition. DO = dispersed oil, C = cascade. with seawater from 1 m depth in the tanks. In situ incubation of two light and one dark bottle lasted 2-4 h, filtration was done on Millipore AAWP filters (porosity = 0.8 #m) which were rinsed with filtered seawater, put in vials and acidified with I ml of 0.1 N HCI. Aquasol (12 ml) was added 2 h later and counting was done with a scintillationcounter (Beckman LS 5801). Frequency of sampling for 14Cuptake was once or twice weeklydue to the time requirements for this technique; (4) lugol-preserved phytoplankton samples, for taxonomic identification; (5) formalin-preserved water samples, for bacterial counts using the epifluorescence acridine orange technique (Hobbie et al., 1977), and (6) total (particulate + dissolved) oil concentration in seawater, by u.v. absorption at 233 nm of dichloromethane extracts. RESULTS
Chlorophyll measurements and taxonomic observations
In the April experiment, strong growth of phytoplankton took place in the five tanks before addition of the dispersed oil on day 0 (Fig. 3). Circulating seawater between the tanks before the beginning of the experiment resulted in relative inter-tank homogeneity in chlorophyll a concentration (cf. day - 1, Fig. 3). After addition of dispersed oil in the three experimental tanks, divergence in the chlorophyll a concentration was soon observed: the DO-100 ( = dispersed oil - 100 ppm) tank showed an immediate leveling off of the chlorophyll a concentration. The decrease in total oil concentration in that tank, down to about 10 ppm after 12 days (Fig. 2), had no effect on the chlorophyll a concentration which continually declined until the end of the experiment. This decline was accompanied by a loss in all previously WR
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Fig. 4. Daily changes in the specific photosynthetic rate (P/B), chlorophyll a concentration (CHL) and DCMUfluorescence increase (Fd-F.), expressed as percent value of controls for the April experiment. Values above 100% (i.e. identical to controls) indicate a stimulation; those below 100% show an inhibition. dominant phytoplankton species (mostly diatoms). The DO-5 (--5 ppm) tank is the only contaminated tank where chlorophyll = concentrations generally remained between those of the two control tanks. Controls diverged in terms of chlorophyll a concentration 4 days after the beginning of the experiment. This may be related to a change in composition of the dominant algal populations after day 4. Diatoms gave way to small unidentified flagellates ( < 10#m) after day 4 in the CTRL-2 ( = control No. 2) tank. Since we had no reason to consider results from either control tanks unreliable, control values were calculated from the mean of results from the 2 control tanks. The 3 experimental tanks were then compared to these control values and a percent control was calculated as the ratio between values obtained from each experimental tank and the means of the 2 control tanks (Fig. 4). In the December experiment, chlorophyll a concentrations were much lower than in the April experiment (Fig. 3). Before the start of the experiment, concentrations varied between 0.5 and 0.8 gg chl a . l-1. This was associated with a very low cell concentration of phytoplankton, the dominant algal populations being micro-flagellates. During the first week after introduction of the dispersed oil mxture, the tank initially contaminated (tank C-h head of cascade) and the
SUZANNE ROY et al.
1252
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In the April e x p e r i m e n t , specific p h o t o s y n t h e t i c rate ( = c a r b o n u p t a k e p e r unit c h l o r o p h y l l a ) gave values a b o v e control, especially for the m o s t c o n t a m i n ated t a n k 1 week after c o n t a m i n a t i o n (Fig. 4 a n d Table 1). S t i m u l a t i o n o f the p h o t o s y n t h e t i c activity c o n t r a s t s with t h e previously d e s c r i b e d i n h i b i t i o n o f c h l o r o p h y l l b i o m a s s (Fig. 3). A c t u a l r a d i o c a r b o n uptake values were still 16.6 m g C m -3 h -], 8 days after c o n t a m i n a t i o n , while chlorophyll a c o n c e n t r a t i o n h a d declined f r o m 37.3 o n d a y 0 to 8.8 m g m -3 (Table 1). A s in the A p r i l e x p e r i m e n t , specific p h o t o s y n t h e t i c rates o f the D e c e m b e r e x p e r i m e n t also s h o w e d values a b o v e control, especially for t a n k s C-1 a n d C-2, the t w o m o s t c o n t a m i n a t e d t a n k s (Fig. 5). This stimulation o f the p h o t o s y n t h e t i c activity again was n o t m a t c h e d by the c h l o r o p h y l l d a t a (Fig. 3).
DCMU-fluorescence In the A p r i l e x p e r i m e n t , D C M U - f l u o r e s c e n c e s h o w e d a d e c r e a s e in p h o t o s y n t h e t i c ability relative to c o n t r o l s in t a n k s D O - 1 0 0 a n d D O - 2 0 following c o n t a m i n a t i o n (Fig. 4). D C M U - f l u o r e s c e n c e generally f o l l o w e d the s a m e t r e n d as the c h l o r o p h y l l a c o n c e n tration. In the D O - 1 0 0 t a n k , D C M U - f l u o r e s c e n c e generally r e m a i n e d b e l o w 2 0 % o f c o n t r o l values, while at the s a m e time, a c c o r d i n g to the r a d i o c a r b o n u p t a k e m e t h o d , p h y t o p l a n k t o n p h o t o s y n t h e s i s was stimulated. In t h e D e c e m b e r e x p e r i m e n t , D C M U - f l u o r e s c e n c e s h o w e d a decrease relative to c o n t r o l s in the cont a m i n a t e d t a n k s C-1 a n d C-2 (Fig. 5) a n d this t r e n d generally f o l l o w e d t h a t o f the c h l o r o p h y l l a c o n c e n tration. In the initially c o n t a m i n a t e d t a n k ( C - l ) , D C M U - f l u o r e s c e n c e values d e c r e a s e d to zero 1 d a y after i n t r o d u c t i o n o f t h e o i l - d i s p e r s a n t m i x t u r e a n d r e m a i n e d at this value until d a y 8. In c o n t r a s t , r a d i o -
Table I. Primary production data. Only the most contaminated tank and control tanks are shown April experiment: Days after contamination Tank PP-light PP-dark D/L B P/B 2 DO-100 85.42 __.3.98 37.40 44% 31.80 + 3.18 2.69 + 0.40 CTRL 216.84 _ 24.66 39.91 + 0.91 18% 88.42 + 2.14 2.45 _+0.34 8 DO-100 16.62 + 0.45 2.31 14% 8.77 ___0.88 1.90 + 0.24 CTRL 57.47+13.12 0.76+0.40 1% 110.78_+91.25 0.71_+0.47 15 DO-100 1.08 _+0.02 0.53 49% 1.09 + 0.11 0.99 -+ 0.12 CTRL 60.66 + 62.11 0.49 -+ 0.57 1% 44.25 ___55.2.8 2.40 _+ 1.62 December experiment: Days after contamination Tank PP-light PP-dark D/L B P/B 1 C-I 0.21 + 0.08 0.20 95% 0.11 1.90 + 0.74 CTRL 0.15 + 0.10 0.13 87% 0.34 0.24 + 0.27 4 C-I 0.65 -+_0.51 0.70 108% 0.09 7.22 -+ 5.65 CTRL 0.19 + 0.10 0 0% 0.23 0.83 + 0.44 6 C-1 0.04 + 0.002 0 0% 0 0 CTRL 0.05 + 0.004 0 0% 0.12 + 0.16 0.42 + 0.63 8 C-I 0.10 + 0.02 0.09 87% 0.11 + 0.07 0.88 + 0.73 CTRL 0.02 + 0.003 0 0% 0.09 + 0.10 0.25 + 0.31 11 C-I 0.09 + 0.03 0.19 210% 0.20 _+0.05 0.45 + 0.26 CTRL 0.06 + 0.03 0 0% 0.10 + 0.01 0.61 _ 0.37 PP-light ffiprimary production (mg C. m3.h- ~) in the light bottles; PP-dark = results from the dark bottles; D/L = percent ratio of PP-light/PP-dark; B = chlorophyll a concentration (mg.m-~); P/B = specific photosynthetic rate (rag C. mg Chl- '. h -' ); value for the controls (CRTL) is the average of the P/B of the 2 control tanks. When replicate measurements were made (as in the 2 light bottles for PP), average values are indicated _+SD.
DCMU-fluorescence and l'C-uptake in oiled reservoirs carbon uptake showed a stimulation of photosynthetic activity relative to controls during this period. DISCUSSION
Techniques comparison The two techniques used here to evaluate phytoplankton photosynthetic activity do not, in fact, measure the same thing: the radiocarbon uptake method gives a value of the rate of carbon uptake on a time scale of hours, while the DCMU-fluorescence method gives information on the pool size of active photosystem II reaction centers (Falkowski and Kiefer, 1985). DCMU-fluorescence reflects the photosynthetic ability of the cell (Vincent, 1980) by expressing the potential for photochemistry and it can be useful as a fast and simple method to indicate inhibition of photosynthesis in cases of contamination (Cullen et al., 1986). In this work, it is surprising that photosynthetic ability (DCMU-fluorescence) seems to be inhibited while photosynthetic rate is stimulated, particularly considering the high concentration of oil used. Closer examination of the data (Table 1) shows that, while both photosynthetic rate (P) and chlorophyll concentration (B) are generally reduced after the oil treatment, the specific photosynthetic rate ( P / B ) is often higher than controls because the reduction in P is generally less than that in B. Highe r than expected 14C uptake values could have resulted in the oiled samples if heterotrophic activity was strong. This is in fact observed here: uptake measured in dark-incubated bottles (Parsons et al., 1984) is quite high (Table 1) and this has been noted in other oil studies (Cross, 1982; Dahl et al., 1983). A bacterial increase following oil contamination likely contributed to this heterotrophic uptake, particularly in the April experiment (Fig. 6). In addition, dispersed oil droplets of 2080 #m size (Coulter particle size spectra, data not presented) were also observed in significant concentralOOO- '
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1253
tion in the oiled tanks and may have contributed to non-algal uptake of the radioactive tracer by physical adsorption on the droplets and retention on the 0.8 #m filters (not actually verified). These effects may be particularly important when the concentration of photosynthetic material is low, as in the December experiment. DCMU-fluorescence on the other hand, was not affected by either the presence of bacteria or dispersed oil particles or by the low concentration of algae present. Replicability of measurements was better (coefficient of variation less than 10%) than with the radiocarbon uptake method (Table 1) even in highly oligotrophic conditions. The dispersed oil fluoresces at a low level under the conditions used for measuring chlorophyll fluorescence in algae (blanks below 0.5 relative fluorescence units) and measurement of the increase in fluorescence after addition of DCMU effectively removes any background effects. This technique has been used successfully in a few other studies dealing with oil contamination (Ostgaard et al., 1984; Siron et al., 1991), with copper toxicity (Cullen et al., 1986) and in mesocosm studies (Keller, 1987). In comparing the two methods used in this study to assess dispersed oil effects on phytoplankton photosynthesis, it appears that the DCMU-fluorescence method is more appropriate and convenient than the radiocarbon uptake method on the following grounds: (1) it is less time-consuming, (2) it does not require the use of radioactive substances, (3) the equipment needed is not expensive and is easy to use in the field, (4) it is not affected by heterotrophic activity or the presence of oil particles, and (5) replicability of measurements is high. Effects of dispersed oil addition on phytoplankton In the April experiment, comparable to a spring bloom condition, dispersed oil introduced in seawater at a concentration close to 10mg.l -~ caused a net decline in chlorophyll a and cell biomass, as well as in the photosynthetic ability of natural phytoplankton enclosed in mesocosms. In the static mode used in this experiment, the effects of contamination lasted as long as the experiment, i.e. there were no obvious signs of recovery. In the December experiment, the actual concentration of total oil in seawater was much lower: this was probably related to a decreased efficiency of the dispersant used in very cold waters and to rapid sedimentation of the added oil before dispersion could act (Siron et al., in preparation). Nonetheless, with oil concentrations around 4 mg. 1-i (tank C-1), a decrease in chlorophyll a biomass and in photosynthetic ability was observed. Although the decrease in chlorophyll biomass was not very distinct from the variations found in the other tanks, probably due to the very low values which are normal for this time of the year in the natural environment (Therriault and Levasseur, 1985), the inhibition of photosynthetic ability in tank C-1 was very clear (Fig. 5). In this December experiment, where mesocosms were run in the cascade
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mode, some signs of recuperation from contamination were detectable after 1 week in the most contaminated tank (Fig. 5), suggesting that residual oil concentrations were not lethal after that time and that dilution can contribute to alleviate toxic effects from dispersed oil. These responses to oil addition are consistent with findings from previous studies run under comparable conditions, i.e. with natural phytoplankton and under field conditions. Concentrations of crude oil-seawater dispersion of the order of 10mg.1-1 and less have been shown to cause deleterious effects on biomass and photosynthetic activity of natural phytoplankton, both in non-renewed large enclosures (Harrison et al., 1986) and in flow-through systems (Linden et al., 1985). As already shown for arctic marine phytoplankton (Hsiao et al., 1978), the work presented here indicates that natural assemblages of phytoplankton from north-temperate environments, such as the St Lawrence estuary in mid-winter or early spring periods, can suffer from contamination with chemically-dispersed oil at relatively low concentrations and that both biomass and photosynthetic activity can be affected. Acknowledgements--The authors wish to thank C. Brochu, L. Bernier, N. Blouin and F. Btlanger for technical assistance. This research was funded by the National Sciences and Engineering Research Council of Canada through its strategic grant program (grant No. STRGP 036). This publication is a contribution of the Oceanographic Centre of Rimouski--a partnership of INRS (Institut National de la Recherche Scientifique) and UQAR (Universit6 du Quebec ~ Rimouski) operating under the auspices of the University of Qutbec.
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