Effects of the concentrations of toxic Microcystis aeruginosa and an alternative food on the survival of Daphnia pulex

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1 424 Notes dation: An experimental test of the size-efficiency hypothesis. Ecology 55: GLIWICZ, Z. M., AND W. LAMPERT Body-size related survival of cladocerans in a trophic gradient: An enclosure study. Arch. Hydrobiol. 129: l-23. -, AND M. G. ROWAN Survival of Cyclops abyssorum tatricus (Copepoda, Crustacea) in alpine lakes stocked with planktivorous fish. Limnol. Oceanogr. 29: , AND H. STIBOR Egg predation by copepods in Daphnia brood cavities. Oecologia 95: HALL, D. J., S. T. THRELKELD, C. W. BURNS, AND P. H. CROWLEY The size-efficiency hypothesis and the size structure of zooplankton communities. Annu. Rev. Ecol. Syst. 7: KERFOOT, W. C Implications of copepod predation. Limnol. Oceanogr. 22: , AND A. SIH Predation: Direct and indirect impacts on aquatic communities. New England. LAZARRO, X A review of planktivorous fishes: Their evolution, feeding behaviors, selectivities and impacts. Hydrobiologia 146: LI, J. L., AND H. W. LI Species-specific factors affecting predator-prey interactions of the copepod Acanthocyclops vernalis with its natural prey. Limnol. Oceanogr. 24: MCQUEEN, D. J Reduction of zooplankton standing stocks by predaceous Cyclops bicuspidatus tho- masi in Marion Lake, British Columbia. J. Fish. Res. Bd. Can. 26: SANTER, B Lebenszyklustrategien cyclopoider Copepoden. Ph.D. thesis, Kiel Univ. 224 p. SMYLY, W. J. P Observations on rate of development, longevity and fecundity of Acanthocycfops viridis (Jut-me) (Copepoda, Cyclopoida) in relation to type of prey. Crustaceana 18: 2 l-36. STEMBERGER, R. S., AND M. S. EVANS Rotifer seasonal succession and copepod predation in Lake Michigan. J. Great Lakes Res. 10: STICH, H. B., AND W. LAMPERT Growth and reproduction of migrating and non-migrating Daphnia species under simulated food and temperature conditions of diurnal vertical migration. Oecologia 61: WILLIAMSON, C. E Behavioral interactions between a cyclopoid copepod predator and its prey. J. Plankton Res. 5: 70 l The swimming and feeding behavior of M~socyclops. Hydrobiologia 134: 1 l- 19. ZARET, T. M Predation and freshwater communities. Yale. Submitted: 22 April I993 Accepted: 17 August 1993 Amended: 10 November 1993 Lmnol. Oceanogr., 39(2), 1994, ,bythe American Society of Limnology and Oceanography, Inc Effects of the concentrations of toxic Microcystis aeruginosa and an alternative food on the survival of Daphnia pulex Abstract-We investigated the effect of the cyanobacterium Microcystis aeruginosa (strain PCC7820) on the survival of juvenile and adult Daphnia pulex (Cladocera) in different food concentrations (the green alga Scenedesmus obtusiusculus). M. aeruginosa reduced survival in D. pulex. The peptide toxin microcystin-lr is present in this strain of M. aeruginosa. The toxic effect decreased with increasing concentrations of S. obtusiusculus. Juvenile D. pulex generally died faster than adults at high concentrations of cyanobacteria. However, juvenile D. pulex did better than adults at the lowest concentration of S. obtusiusculus. Laboratory experiments focusing on the potential toxicity of cyanobacteria to planktonic crustaceans have produced some contradictory results (see Lampert 1987; Paerl 1988; de Bernardi and Giussani 1990). For instance, in a study by de Bernardi et al. (198 1) all three cladoceran species tested were able to grow and reproduce on diets consisting exclusively of Microcystis aeruginosa; Lampert (1982), on the other hand, found that 13 cladoceran species were negatively affected by this cyanobacterial species. Other studies have compared several cyanobacterial strains and have shown that some strains are toxic to cladocerans, whereas others are not (Nizan et al. 1986; Vasconcelos 1990). In recent years, the chemical composition of cyanobacteria has been intensively studied (e.g. see Carmichael et al. 1990). However, only a few studies involving cyanobacteria and zooplankton have included toxin analyses (Lind-

2 Notes 425 holm et al. 1992). DeMott et al. (199 1) showed acute toxic effects of two cyclic peptides (a microcystin and nodularin) from cyanobacteria on Diaptomus and Daphnia. Jungmann (1992) fractionated an extract of M. aeruginosa and found lethal effects on Daphnia in a fraction that did not contain microcystins but did include one or more unidentified compounds. These examples with purified and partly purified compounds show that cyanobacteria may contain several substances, some unidentified, that are toxic to crustaceans. Quantitative differences in the toxicity of cyanobacteria to crustaceans may also derive from varying experimental conditions, such as the amount of food present in cyanobacterial exposures. We hypothesize that when less food is available the toxic effects will be greater. We also hypothesize that different life stages may respond differently. In natural waters, declines in populations of large Daphnia are sometimes associated with cyanobacterial blooms (Edmondson and Litt 1982; Infante and Abella 1985; Jarvis et al. 1987) indicating that cyanobacteria may affect interspecies competition in zooplankton. The varying responses of different species may result from both differential physiological sensitivity and behavioral responses such as feeding behavior (DeMott et al ). Feeding behavior is often dependent on resource availability (e.g. DeMott and Moxter 1991). Furthermore, laboratory studies using cladocerans of different sizes and ages (Lampert 198 la; Nizan et al. 1986; Gilbert 1990) suggest that these factors may also be important. The purpose of this study is to test the acute toxicity of M. aeruginosa strain PCC7820 to Daphnia pulex subjected to different food concentrations (Scenedesmus obtusiusculus). We focused on ways in which the toxic effects of A4. aeruginosa may be altered by different food concentrations and by D. pulex life stage. The M. aeruginosa strain has been used in many toxicological studies on vertebrates, and it has been shown that its toxicity to vertebrates depends mainly on the production of the heptapeptide microcystin-lr (Carmichael et al. 1990). The D. pulex clone used was originally isolated from a eutrophic pond in Turku (SW Finland) in We have previously studied the life-history traits characteristic of this clone (Ketola and Vuorinen 1989; Walls and Ketola 1989; Vuorinen et al. 1989; Walls et al ). The experimental stocks were grown in GF/C filtered water from Littoistenjarvi, a lake near Turku. The filtered lake water has been used extensively in our laboratory with no known side effects. Our culture of the unicellular M. aeruginosa strain PCC7820 was grown in a 28 medium (Kotai 1972). The experimental culture was prepared by diluting a stock culture to 4 x lo6 cells ml- l3 d before beginning the experiment. The culture was thereafter diluted to this concentration daily. It was grown at 25 C under continuous illumination (N 20 PEinst mm2 s- l). The cells were harvested by passing a subsample of the culture through a Whatman GF/C filter. Thereafter the cells were resuspended in Whatman GFK-filtered lake water. Stock and experimental cultures of S. obtusiusculus were grown in a nutrient medium (Kylin et al. 1972) and kept constantly in an exponential growth phase. For the toxin analysis, a 5-ml subsample was taken from the cyanobacterial culture three times during the experiment. The subsample was passed through a Whatman GFK filter; the filter was deep frozen (- 20 C) and freezedried. For extracting and analyzing microcystin-lr, we followed a modification of the internal surface reversed-phase HPLC method described by Meriluoto et al. (1990a,b). The filters were extracted twice with 5 min of ultrasonication and 1.O ml of 10% acetonitrile- 90% 0.1 M KH2P04, ph 2 (low ph extraction has been described by Harada et al. 1988). The sample was centrifuged for 5 min at 4,000 x g after each extraction and the supernatant collected. The combined supematants were then passed through Gelman Acre LC ~pm filters. Twenty microliters were injected into the HPLC system. The column was a Regis 250- x 4.6-mm GFF-SS-80 column. It was eluted at 1.O ml min-l with 15% acetonitrile- 85% 0.1 M KH,PO,, ph 6.8. Peak identification was further ensured with 10% acetonitrile-90% 0.1 M KH2P04, ph 2. The signal was read at 238 nm. The experiment was carried out as follows: each daphnid was reared individually in a 20- ml vessel at 20 C and a 16 : 8 L/D cycle. The animals were transferred daily into fresh treatment media (i.e. we used a semistatic system).

3 426 Notes The treatments included seven concentrations of cyanobacteria (0 for control and 2,500, 6,000, 14,000, 32,000, 70,000, and 160,000 cells ml-l) and four concentrations of S. obtusiusculus (0, 10,000, 20,000, and 100,000 cells ml-l). A food concentration of 10,000 cells ml-l is about the lowest at which our D. pulex clone reproduces in life-cycle experiments when a 20-ml vessel is used (pers. obs.). The treatments were carried out with both juveniles (under 24 h of age at the beginning of the exposure) and adults (egg-bearing individuals). There were seven replicates of each treatment combination. The time of exposure was 4 d (96 h). Adult animals were collected directly from the mass cultures. Juveniles were collected as follows: 24 h before the start of the experiment, females were transferred from the mass culture into 600-ml vessels with similar conditions as in the mass culture. Newborn daphnids were collected the next day. Both adult and juvenile Daphnia were randomly assigned to the different treatments. There was only small variation in the sizes of both life stages: the average body length of the adults was 2.30 mm (SE, 0.07) and that of the juveniles 0.67 mm (SE, 0.02), measured as the distance from the ventral base of the spine to the anterior part of the compound eye. The survival data from the experiment were tested with ANOVA (SAS Inst. 1989). We first ran a three-way ANOVA for the whole set of data, using D. pulex life stage, food level, and cyanobacterial concentration as main factors. When a significant three-factor interaction was found, we compared the mean-square values (MS) of the main factors with the MS of the three-factor interaction. Because the MS of the main factors food level and cyanobacterial concentration were at least one order of magnitude greater than the MS of the three-factor interaction, these main factors were also interpreted (Mead 1988); differences among groups were tested with Tukey s a posterior-i test (Sokal and Rohlf 198 1; SAS Inst. 1989). For the analyses, we calculated the survival time (days) for each animal during the trial. Animals that died during the first 24 h were assigned a survival time of 0 d; animals that did not die during the exposure (96 h) were assigned a survival time of 4 d. The residuals were normally distributed. Nevertheless, since the experiment ended before all the animals died, the variances were not homogeneous, and a nonparametric test would have been appropriate (e.g. see DeMott et al ). However, interactions are of special interest in this study and nonparametric tests are not equipped to measure interactions (a nonparametric test would also have lost power due to a large number of ties in the data), so we decided to use a parametric test. Profiles were also used to interpret the relative importance of the different main factors when interactions were found. Most animals at low cyanobacterial concentrations and in the control treatment would presumably have had longer survival times than the maximum of 4 d if the experiment had not ended. Consequently, the difference in survival of D. pulex in treatment levels that did and did not cause mortality becomes less pronounced than if the experiment had been continued. Any conclusion as to the lethal effect of a treatment combination is therefore an under- rather than an overestimate. LCso values were calculated with PROBIT analysis (Natl. Swedish EPA 1989). The concentration of microcystin-lr in our culture was 0.14 pg ml-l (SE, 0.05; n = 3) at the given concentration of cyanobacteria (-4 x lo6 cells ml- l). With a dry weight of x 10m5 pg cell-l (DeMott et al. 199 l), this is -0.17% of the dry weight of the cells. Even though there was some variation between measurements, the concentrations stayed consistent with typical values reported for this strain (DeMott et al ). The presence of cyanobacteria reduced survivorship in all combinations of life stage and food level (Fig. 1). When food levels were low, the concentration of cyanobacteria causing mortality was lower than when food levels were high. There were also differences in survivorship between the life stages. In the juveniles, survivorship was already drastically reduced after 1 d of exposure, whereas there was almost no mortality in adults at this time. However, after 4 d of exposure, mortality was about the same in both groups, with the notable exception that at the lowest level of food the juveniles did better than the adults. Three-way ANOVA of the survival time data showed a statistically significant S. obtusiusculus x M. aeruginosa x life-stage interaction; the effects of the main factors were also statistically significant (Table 1). The mean sur-

4 Notes 427 Juvenile Scenedesmus 1.0' , Adult 0 cells ml-l Scenedesmus 0 10,000 cells ml-1 Scenedesmus 20,000 cells ml-1 Scenedesmus o I L I I l.ol 100,000 cells ml-l Time (d) 1211 o.o(j Fig. 1. Survivorship of juvenile and adult Daphnia pulex exposed to different concentrations of Microcystis aeruginosa at different food levels (Scenedesmus obtusiusculus). 0 = 0; 1 = 2,500; 2 = 6,000; 3 = 14,000; 4 = 32,000; 5 = 70,000; 6 = 160,000 M. aeruginosa cells ml-l.

5 428 Notes Table 1. Results of a three-way ANOVA testing effects of Microcystis aeruginosa (Ma), Scenedesmus obtusiusculus (So), and Daphnia pulex life stage (LS) on survival (days) of D. pulex. Means not sharing a common solid underline are significantly different (Tukey s a posterior-i test). Subscripts indicate concentrations (cells ml-l) in the So (3 = 100,000; 2 = 20,000; 1 = 10,000; 0 = 0) and Ma treatment levels (6 = 160,000; 5 = 70,000; 4 = 32,000; 3 = 14,000; 2 = 6,000; 1 = 2,500; 0 = 0) and D. pulex life stage in the LS treatment levels (j-juvenile; a-adult). Source df MS F P Means of treatment levels (No. of replicates) SO Ma LS So x Ma so x LS Ma x LS SoxMaxLS Error Mean, Mean, Mean, Mean,, (97) 2.6(97) 2.3(97) 2.2(98) Mean, Means Mean, Mean, Mean* Mean, Mean, (56) ~ 1.2(55) - 1.9(56) 3.0(56) 3.8(56) 3.8(55) 3.9(55) Mean, Mean, (194) - 2.7(195) viva1 times at the three lowest concentrations pulex, which is in accordance with earlier works of cyanobacteria were significantly longer than by Lampert (198 1 a, b), Nizan et al. (1986) and at the higher concentrations. Furthermore, the DeMott et al. (199 1). For instance, 14,000- mean survival times at the four highest con- 32,000 M. aeruginosa cells ml- l drastically recentrations of cyanobacteria differed signifi- duced survivorship at the low food levels (Ocantly from each other (Table 1). 20,000 S. obtusiusculus cells ml- ; Fig. 1). The mean survival times at the two lowest It is of interest to compare our findings to levels of food were significantly shorter than the work of DeMott et al. (199 l), who used at the two highest levels. Furthermore, the purified toxin as well as intact M. aeruginosa mean survival times at the two highest levels cells (strain PCC7820), so we have calculated of food differed significantly from each other the 48-h LCsO value (cyanobacterial cells) for (Table 1). Figure 2A also shows that increased the treatments involving 10,000 S. obtusiuslevels of food enhanced survival. culus cells ml-l (adult D. pulex). These con- Juvenile and adult D. pulex reacted differ- ditions correspond approximately to those used ently. Juvenile mortality was 49% during the by DeMott et al. The 48-h LCsO value is 30,000 first day and 45% during the second day. For cells ml-l (95% C.I. = 15,800-86,000 cells adults, the corresponding values were 3% and ml-l). The microcystin-lr content in this 59%. Adult D. pulex consequently had longer number of cells, according to our HPLC analmean survival times than the juveniles, but ysis, is only pg. DeMott et al. reported the MS of the life-stage treatment was of the that the 48-h LCsO value for purified, dissolved same order of magnitude as the MS of the microcystin-lr was 9.6 pg ml-l for D. pulex. three-factor interaction (Table 1); conclusions There is a difference of almost four orders of as to the effect of life stages should therefore magnitude between these estimates. DeMott be drawn with caution. et al. suggested similar relationships between The effect of life stage varied depending on the toxicity of dissolved and cellular microthe concentration of cyanobacteria and the lev- cystin-lr to daphnids: they calculated the ap- el of food. Adult D. pulex had slightly shorter survival times than juveniles at the lowest cyanobacterial concentrations but did better than the juveniles at the highest concentrations (Fig. 2C). Juvenile D. pulex survived slightly better than adults at the lowest food level but had shorter mean survival times at the higher levels of food (Fig. 2B). We conclude that low amounts of M. aeruginosa are sufficient to cause mortality in D. proximate dry weight of the cyanobacteria needed to produce an amount of purified toxin having a lethal effect and compared this to the dry weight of intact cells that has a similar effect (10 mg ml- and 0.2 pg ml-l respec- tively). This difference was also four orders of magnitude. Why are cyanobacterial cells so much more toxic than the equivalent amount of dissolved microcystin-lr? One explanation is that some

6 Notes 429 other substance(s) in the cyanobacterial cells is (are) more toxic than microcystin-lr. Studies by Nizan et al. (1986) and Vasconcelos (1990) have shown that cyanobacterial strains not considered toxic to mice (i.e. not containing peptide toxins for instance) may be toxic to some crustacean zooplankters. This conclusion gained support in a study by Jungmann (1992): a fraction of a M. aeruginosa extract was lethally toxic to Daphnia pulicaria, although it did not contain microcystins. Another fraction contained microcystins but not in sufficient amounts to cause mortality. However, in the study by DeMott et al. (199 l), the toxicity of purified and dissolved microcystin- LR corresponded closely to the toxicity of a dissolved crude extract from the cyanobacteria that contained microcystin-lr as well as other substances. This result suggests that microcystin-lr was responsible for the toxic action but that it lost most of its potential when it was delivered via water rather than via food. Differences in toxicity due to different modes of delivery have been intensively studied in aquatic toxicology (e.g. Moriarty 199 1). Future studies on the structure and toxicity of the new compound(s) suggested by Jungmann (1992) may reveal more about the relative role of the different substances present in cyanobacteria. Furthermore, the possible synergistic effects among the different toxins present in cyanobacteria need attention. It is important to note that factors other than the toxin concentration itself may have considerable influence on the results of acute toxicity tests; this possibility is demonstrated in our work by the use of several concentrations of food. Obviously, cyanobacterial concentrations that are lethal at a low concentration of food may have no effect on survival at a higher food concentration. For instance, 14,000 M. aeruginosa cells ml- l (corresponding to pug microcystin-lr ml-l) killed all the adults at 0 S. obtusiusculus cells ml- l, but when the food level was 100,000 S. obtusiusculus cells ml-l, a concentration of 160,000 M. aeruginosa cells ml-l ( pg microcystin- LR ml-l) was required to produce this effect (Fig. 1; see also the plot in Fig. 2A for 32,000 M. aeruginosa cells ml- ). These observations reflect the role of food concentration and cyanobacteria in the significant S. obtusiusculus x M. aeruginosa x life-stage interaction (Table 1). 0 A I I,, I,, 50 80,, 70,, 80,, 90,, c 100 Scenedesmus (cells ml-1 x 1,000) Scenedesmus (cells ml-l x 1,000) Microcystis (cells ml-l x 1,000) Fig. 2. Survival (&SE) of two life stages of Daphnia pulex exposed to different concentrations of Microcystis aeruginosa and Scenedesmus obtusiusculus. A. Data for adult and juvenile animals were pooled, and survival at different concentrations of M. aeruginosa was plotted against food level. 0 = 0; 1 = 2,500; 2 = 6,000; 3 = 14,000; 4 = 32,000; 5 = 70,000; 6 = 160,000 M. aeruginosa cells ml- I. B. Data for the different M. aeruginosa concentrations were pooled and plotted against food level. C. Data for the different food levels were pooled and plotted against M. aeruginosa concentration. In panels B and C, dashed lines indicate adult D. pulex and solid lines represent juveniles. Lampert ( a, b) also used different concentrations of M. aeruginosa combined with one Scenedesmus concentration each. By using four Scenedesmus concentrations per each cy-

7 430 Notes anobacterial concentration, we were able to demonstrate the interactive effects of cyanobacterial exposure and an alternative food on the survival of two life stages of D. pulex. We have considered several explanations for the interaction of food level and cyanobacteria. First, at low concentrations of food, a greater proportion of the total amount of food is toxic, increasing the probability that the daphnids will ingest toxic particles. Second, the daphnids might selectively discard food particles of low quality (M. aeruginosa) directly upon capture when food of high quality is abundant. This hypothesis is inconsistent with studies that have shown how poorly daphnids discriminate against food particles of low quality (e.g. Richman and Dodson 1983; DeMott and Moxter 199 1). Third, postcapture rejection of food from the filter chamber by means of abreptor and labrum movements may occur more often when better food is abundant. Mechanisms for food rejection have been described by Burns (1968), Porter and Orcutt (1980), Porter et al. (1982), and Richman and Dodson (1983). Fourth, the daphnids are physiologically better off when food of high quality is abundant and may therefore be more tolerant to the toxin. A more thorough discussion of this subject would require data on the feeding behavior of the animals during the experiment. We lack these data. Nevertheless, food concentration can be expected to heavily impact the effect of toxic cyanobacteria on crustaceans. The importance of the life stage of the animals is also interesting. Almost all (93%) of the experimental animals that died during the first 24 h were juveniles. Even though juveniles reacted faster to the toxin(s) than adults, resulting in shorter mean survival times, there was virtually no difference in survivorship when the whole time of exposure was taken into account; 54% of the animals that did not die during the exposure were juveniles. At the lowest level of food, survivorship was even higher for the juveniles, and most (70%) of the animals that did not die during the experiment were juveniles. This observation may mean that the maternal lipids of the juveniles make them physiologically more tolerant to the toxin(s) when there is no high-quality food present or provide energy if the animals cease feeding completely. According to our observations, maternal lipids are present in newborn D. pulex for at least 2-4 d. We conclude that juveniles are more sensitive than adults to high levels of M. aeruginosa, but they are equally or less sensitive to intermediate concentrations. This conclusion is supported by the profiles in Fig. 2C. We also conclude that juveniles survive better than or equally as well as adults when no high-quality food is added. This conclusion is supported by the profiles shown in Fig. 2B. These observations reflect the role of life stage in the statistically significant three-factor interaction among food level, cyanobacterial exposure, and life stage (Table 1). In planning acute toxicity tests with Daphnia and cyanobacteria, the differences between life stages (and possibly animals of different size) and the importance of the time of exposure and food levels should be considered. Differences between life stages are reduced when sufficiently long exposure times are used. Our results show that planktonic cyanobacteria can be expected to regulate the population dynamics of crustacean zooplankters in a complex way. D. pulex is a species that will only occasionally be exposed to mass occurrences of cyanobacteria because it inhabits mainly small ponds. Such a species may be severely suppressed by cyanobacteria, since both its behavioral and physiological tolerances to cyanobacteria are low (DeMott et al ; but see Andersson and Cronberg 1984). The magnitude of the suppression depends on the toxin(s) involved and also on resource availability. Furthermore, the impact of food availability and cyanobacteria on D. pulex populations depends on the age structure of the populations. Department of Biology University of Turku FIN Turku, Finland and Department of Biology Abe Akademi University Biocity FIN Turku Department of Biology University of Turku Marko Reinikainen Matti Ketola Mar-i Walls

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9 432 Notes WALLS, M., H. CASWELL, AND M. KETOLA De- duced spines on individual fitness in Daphnia pulex. mographic costs of Chaoborus-induced defences in Limnol. Oceanogr. 34: Daphnia pulex: A sensitivity analysis. Oecologia 87: Submitted: 25 January Accepted: 23 March , AND M. KETOLA Effects of predator-in- Amended: 14 December 1993 LlrntlOl. Oceanogr., 39(2). 1994, (0 1994, by the Amencan Society of Limnology and Oceanography, Inc. Has the importance of photoautotrophic picoplankton been overestimated? Abstract - Postincubation differential filtration (PIDF), preincubation differential filtration (Pre- IDF), and track autoradiography (TA) were compared for estimating cell-specific and total photoautotrophic picoplankton production. Experiments were performed in Lakes Michigan and Huron and in the Gulf of Mexico. When Synechococcus dominated the photoautotrophic picoplankton community (> 70% of total picoplankton abundance), PIDF estimates of cell-specific and total picoplankton production were -3.0 x (range, X) higher than TA estimates. PreIDF estimates of cell-specific and total picoplankton production, however, were only slightly higher than TA estimates (mean, 1.4 x ; range, 1.4-l.5 x ). The higher PIDF estimates were attributable to breakage and damage of larger photoautotrophs during postincubation filtration and to retention of this labeled material on the smaller (0.2 Fm) pore-size filter. Results from PIDF experiments must be viewed with caution and previous estimates of picoplankton production, cell-specific or total, based solely on PIDF may need to be re-evaluated. Research during the past decade in a wide variety of environments (see Stockner and Antia 1986) has demonstrated the importance of photoautotrophic picoplankton as significant contributors to primary production and photoautotrophic biomass. Photoautotrophic picoplankton can contribute as much as 90% to total primary production (Li et al. 1983; Itur- A&nowledgments We thank two anonymous reviewers for comments on the manuscript. Technical assistance and discussions were provided by Hunter Carrick. This work was supported by a grant from the Coastal Ocean Program, Nutrient-Enhanced Coastal Ocean Productivity Program (NOAA). GLERL contribution 868 and CMSKJSM contribution 172. riaga and Marra 1988). Many of these estimates of picoplankton production - particularly the very high estimates- have been made with postincubation differential filtration (PIDF) experiments (Glover et al. 1986; Iturriaga and Mitchell 1986; Hagstrom et al. 1988; Iturriaga and Marra 1988). Likewise, much of the rate process information for Synechococcus and other picoplankton-sized organisms has been made with PIDF experiments (Iturriaga and Mitchell 1986; Prezelin et al. 1986). Preincubation differential filtration (PreIDF) has been used to estimate picoplankton production (Waterbury et al. 1986) but because this technique may introduce possible artifacts it has been viewed with caution (Fumas 1987). The accuracy of PIDF for estimating picoplankton production has been questioned. Waterbury et al. (1986) noted that the percent of primary production attributed to picoplankton often exceeded 100% and could be as much as 175% of total primary production if the PIDF protocol were used. They attributed these biased estimates of picoplankton production to disruption of eucaryotic phytoplankton during filtration and to retention of cellular fragments on the small pore-size filters. Also, Iturriaga and Marra (1988) noted that 14C-based growth rates from PIDF experiments were about twofold higher than estimates from track autoradiography (TA). Although these studies questioned the accuracy of PIDF procedures for estimating picoplankton production, they provided only a cursory evaluation of the PIDF technique. In this note, we build on the work of Waterbury et al. (1986) and Iturriaga and Marra (1988)