FINAL REPORT: NOAA PUERTO RICO PHARMACEUTICAL WASTES c. Van Baalen, University of Texas Marine Science Institute, Port Aransas Marine Laboratory, March 15, 1981 The proposed work included a re-test of individual pharmaceutical wastes from Puerto Rico to see if the pattern of algal toxicity ·found in the studies in 1979 would repeat in a new set of samples collected in 1980. In addition, Bristol waste, highly toxic in the 1979 and again in the 1980 algal bioassays, was subjected to purging or exposure to sunlight to gauge if toxicity was perhaps volatile or photosensitive. Bristol and Upjohn (the second most toxic waste) wastes were also examined for any immediate effects on photosynthesis with organism PR-6 . Six of the individual wastes were also mix~d in approximab~ly the same proportions as they occurred in a composite waste, and bioassayed with the marine blue-green alga, PR-6, to ascertain the waste or wastes contributing the most algal toxicity to composite samples. MATERIALS AND METHODS The principle test organism used was the marine, coccoid, blue-green alga, Agmenellum quadruplicatum, strain PR-6 (Van Baalen, 1962), the diatom, Cylindrotheca sp. strain N-1 (our isolate) and Chlorella autotrophica, strain 580 (R.R.L. Guillard) were also used. The growth medium was a modification of the original formula (Provasoli, et al., 1957; Van Baalen, 1962) used either at full strength or diluted as appropriate with sea water. The ASP-2 medium may be diluted in half with distilled water or sea water without appreciable effect on the growth of organism PR-6. The untreated wastes were added directly to the growth mediu,~, v/v, just before inoculation and incubation, see Figure 1 for details. Growth rates and lag times in initiation of growth as compared to suitable controls were the experimental endpo_ints. A typical set of growth curves is shown in Figure 2. The composite samples, identified as Mix 1 and 2 (Table 5) were purged by passing tank N2 through 3ml of sample in a sterile 7ml tube for 24 hours in darkness at room temperature. The "Barge" sample was received as separate purged and unpurged samples (Brooks, Texas A&M) . The purged and unpurged samples were then added directly to screw-cap 22xl75 test tubes, medium added, the tubes inoculated, and then incubated in a growth bath. Carbon dioxide was periodically added through a small hole in the screw cap backed by a chromatography septum. The Bristol waste was examined for photosensitivity by adding it directly, v/v, to lOml of filtered (0.2µm Nucleopore), off-shore sea water in a quartz 18xl65mm test tube. The quartz tubes were then placed outside in direct sunlight. Tubes were removed and frozen after 24 hours (approx. 12 hours light and 12 hours dark) or after 72 hours. The samples were then diluted in half with medium ASP-2, inoculum added, and growth rates determined. Photosynthesis measurements were made using a YSI Co., Inc. No. 5331 oxygen electrode in a water-jacketed glass cell (Van Baalen, 1968). A suspension of cells of organism PR-6 containing a given amount of Bristol waste was placed in the electrode chamber, sealed with a glass stopper and after several minutes in the dark to record a respiration rate the light was turned on and the increase in electrode current recorded. RESULTS Table la shows the effects of the 1980 samples of individual pharmaceutical wastes on the growth of organism PR-6 and for comparison, the same data is shown in Table lb for the 1979 wastes. The data are remarkably consistent. In the 1980 samples it was again the Bristol and Upjohn wastes which were inhibitory. The lower inhibitory levels of Bristol and Upjohn were titrated, (Table 2) and it would appear that the 1980 Bristol sample was even more toxic than the 1979 sample. The composite sampl~s, Mix 1 and 2 and "Barge'', were also toxic in the open growth system to .the blue-green alga PR-6 and the diatom N-1, while the green alga, 580, was little affected (Table 3) . Bristol waste was extremely toxic by itself but when mixed in proportion to its typical concentration (3%) in a composite waste then it seemed to exert little effect on toxicity (Table 4). The Upjohn waste, because it constituted nearly 50% of a composite waste, was clearly the cause of the algal toxicity. Omission of the other wastes; Merck, Capri, Pfizer, or Shering had little effect on the overall toxicity. A comparison of the composite samples, Mix 1, 2 and "Barge", purged or unpurged, on growth of organism PR-6 using a closed growth system (screw-cap test tubes with periodic addition of co (Table 5) suggests volatiles have little to 2 do with the toxicity of the composite samples. However, this conclusion is compromised by the slower growth rates in the closed system and volatiles still could be contributing, albeit to a small extent, to the overall toxicity. Figure J · records the immediate effects of Bristol and Upjohn wastes on photosynthesis (0output) in organism PR-6. 2 The curve at the top marked DCMU, a well-known and very potent inhibitor of photosynthesis, shows what might be expected. Neither Bristol or Upjohn wastes at relatively high concentrations (see Table la), mimicked the action of DCMU but the immediate depressions in rate of oxygen evolution of approximately 10% are real and therefore bothersome. This data is taken as an indication that a primary cause of lethality of Bristol and Upjohn wastes may lie iP direct damage to the photosynthetic machinery. Bristol waste, 250ppm or 500ppm, in filtered sea water was exposed for ~p to 3 days to sunlight (approximately 36 hours) and then tested at 125ppm or 250ppm in half strength sea water-half strength medium ASP-2. There was no evidence of a considerably decreased toxicity due to photochemical breakdown. DISCUSSION The repetition of the pattern of toxicity of the 1979 samples using organism PR-6 with the 1980 samples is very close. This strongly suggests that the toxicity of a composite is due to persistent material(s) basic to routine chemical operations rahter than to a "one-shot" situation. There is an interesting discrepancy in the data in Table 4. When Upjohn waste was omitted then the mocked-up composite was non-toxic. However, at 2500ppm the sample contained 75ppm Bristol waste, at 5000ppm, 150ppm, well within the range wherein Bristol toxicity should have been readily -evident (Table 2). It may well be that the toxicity of Bristol waste is, for unknown reasons, not expressed in the presence of something contained in other waste(s). The data (Table 5) do not support the notion that volatile compounds are the basis for the toxicity of composite samples. However, as noted before, the closed algal growth system leaves something to be desired and therefore the results may be compromised. The attempts to show that the toxi~ component(s) in Bristol might be largely photosensitive have also failed to find evidence for this. The experiments on effects of Bristol or Upjohn wastes on short-time photosynthesis in organism PR-6 suggest that there is an immediate, within 5 to 8 minutes after adding waste to the algal suspension, depression in oxygen evolution. The depression is small, 10% or so, but it does argue that the site of toxicity may lie in the photosynthetic machinery. We have no ready recommendations for alleviating the evident toxicity of individual wastes such as Bristol or Upjohn, nor for the composite wastes. The data herein demonstrate the level at which composite pharmaceutical wastes are lethal to representative microalgae when tested at population densities 500 to 1000 or more times natural phytoplankton levels (as chlorophyll a). As is usually the case it is difficult to extrapolate the lab data to the field. However, one thing the lab data do provide is a clean statement that single pharmaceutical wastes can be highly algicidal and that composite wastes, -although they must necessarily be used at higher concentrations, are also algicidal. It may not be cricket to dismiss the algal toxicity of these pharmaceutical wastes simply because the concentrations are assumed to rapidly diminish in the real world in a linear manner. We use high concentrations in the lab in order to clearly see effects on an easy to measure sum of cellular metabolism-growth. Growth rate is a convenient way to gauge a cell's overall happiness with his external milieu. However, there can be appreciable changes in a cell, in basic cellular process such as cell division, which are not reflected in any diminished rate of overall biosynthesis. For example, the growth rate, as dry weight increase, of a filamentous cell division impaired mutant can be the same as the wild-type (Ingram and Van Baalen, 1970). This example illustrates that very considerable changes in a fundamental cellular process­cell division-can occur without effect on rate of dry weight increase. It would be most difficult or impossible to see such an effect in phytoplankton populations in nature. Therefore without some idea of what metabolic site(s) the toxic materials affect it seems well to exercise caution in ascribing "no toxicity" to a waste simply because the concentration levels needed in the lab to see algistatic or algicidal effects are judged unrealistic in the sea. There may well be significant alterations in regulation of biosynthesis leading to drastic changes in cell composition or morphology. Such changes in the phytoplankton can be amplified through the food chain. Table la. Growth rate of organism PR-6, as generations per day, in the presence of individual pharmaceutical wastes, 1980 samples. Growth in open test tubes with continuous illumination and aeration with 1% coin 2 air. Waste Material Concentration Generations Identification ppm per day Controls 0 5.3 + . 3 Bristol 250 NGa 500 NG 2500 NG 5000 NG Up john 250 3.0 (lOO)b 500 NG 2500 NG 5000 NG Merck 250 5.4 500 5.4 2500 5.4 5000 5.2 Squibb 250 5.5 500 5.3 2500 5.3 5000 5.1 Capri 250 5.4 500 5.4 2500 5.4 5000 5.2 Pfizer 250 5.4 500 5.4 2500 5.4 5000 5.4 Shering 250 5.4 500 5.3 2500 5.3 5000 4.9 a NG means no growth for up to 5 days incubation. b .Number in parenthesis is lag time in hours as compared to control. b J.S t in e in s as . Number in par is lag control. in tisis is lars lag compared to parnber in is is t . Table lb. Growth rates, as generations per day, of organisms PR-6, N-1 and 580 in the presence of individual pharmaceutical wastes, 1979 samples. Growth in open test tubes with continuous illumination and aeration with 1% coin air. 2 Waste Material Concentration Organisms Identification ppm PR-6 N-1 580 Controls 0 5. 3+. 3 4.7+.3 2.9+.3 Bristol 250 NGa 2.4 NG 500 NG NG NG 2500 NG NG NG 5000 NG NG NG Upjohn 250 NG 4.5 3.0 500 NG 4.5 3.0 2500 NG 4.0 b 3.0 5000 NG 2.5(26) 3.1 Merck 250 4.9 4.7 3.0 500 4.9 4.5 3.0 2500 4.3 4.6 2.6 5000 2.5 4.6 2.6 Squibb 250 5.3 4.5 2.9 500 5.3 4.5 2.9 2500 4.5 3.5(17) 3.1 5()00 4.1 3.5(24) 3.1 Capri 250 5.4 4.7 2.9 500 5.4 4.7 2.9 2500 5.4 4.6 2.8 5000 5.0 4.6 2.8 Pfizer 250 5.2 4.7 2.8 500 5.1 4.6 2.8 2500 5.1 4.7 2.8 5000 5.1 4.5 2.8 Shering 250 4.4 4.6 2.9 500 4.3 4.6 2.9 2500 2.3 4.6 2.8 5000 1.1 4.7 2.8 aNG means no growth for up to 5 days incubation. b mb . Nu er in parenthesis is lag time in hours as compared to a control. Table 2. Growth rate for organism PR-6, as generations per day, in the presence of Bristol and Upjohn wastes, 1980 samples. Growth in open test tubes with continuous illumination and aeration with 1 % coin air. 2 Waste Material Concentration Generations Identification ppm per day Control 0 5.3 + . 3 Bristol 1. 3 5.0 2.5 2.6 (138)b 6.8 NGa 12.5 NG 25.0 NG 63.0 NG 75.0 NG 150.0 NG 250.0 NG Upjohn 150.0 5.3 (12) 250.0 3.0 (100) 500.0 NG aNG means no growth for up to 10 days incubation. bNumber in parenthesis is lag time in hours as compare