C C NATURAL LEVELS OF DDE AND THE UPTAKE OF C BY DIFFERENT SIZE CLASSES OF THE MUSSEL MYTILUS CALIFORNIANUS (CONRAD, 1837) Karen K. Davis Hopkins Marine Station:" Stanford University -DDT FOOTNOTES 1Present address: This work was supported in part by the Undergraduate Research Participation Program of the National Science Foundation, Grant + G4-5878. O RUNNING HEAD DDT Uptake by ! tilu O O INTRODUCTION Slightly over twenty years ago the introduction of DDT to the world environment was received as the great panacea for the control of insect pests of man's food crops. Furthermore, its success in controlling malaria and other human diseases carried by insects was undeniable. Only recently, however, have studies of the long-term effects of DDT on non-target organisms begun to cause alarm. Concern is growing now over the discovery that although the actual DDT concentration in the oceans is only in the order of 5-15 parts per trillion, DDT is taken up by phytoplankton and then passed along the food chain through zooplankton, fish, and birds where it may reach toxic levels (Risebrough, Reiche, Peakall, Herman, and Kirven 1968; Risebrough, Menzel, Martin, and Olcott 1967; Hickey and Anderson 1968). The organism studied, Mytilus californianus, is a common species of the intertidal region of the Monterey Bay area. As a filter feeder it transports large quantities of water through the mantle cavity. Many suspended nonliving particles and small living organisms also enter with the water, come into contact with the ctenidia and may or may not be picked up by the palps and passed through the alimentary tract. It was sus- pected that DDT might be accumulated from contact with water passing through the mantle cavity as well as from particles of page 2 food passing through the entire digestive system. The varia- bility of background DDT levels occurring in natural populations from different areas, especially in respect to size, was also considered important because these levels could affect further DDT uptake. Furthermore, because of the local variation in pesticide levels, it would be interesting to know to what extent the immediate environment of the animal might affect its concentration of DDT. The aim of this research was to determine background levels of DDT and its residues in natural populations of Mytilus californianus and to compare the uptake of C-labeled DDT by the mussel through different modes of entrance: directly from the surrounding sea water, and from phytoplankton, part of its natural food supply. The results of this study show that DDT does not only enter the food web via the phytoplankton, but may also be taken up in substantial quantities by organisms higher in the food web directly from the surrounding water. MATERIALS AND METHODS The experimental animals were collected in April and May of 1969 from four different localities around the Monterey peninsula. These were Cabgrillo Point, Point Pinos, Point Joe, and Seal Rock in Monterey County, California. The areas were chosen to represent populations from typical bay and outer coast localities. The animals were all collected from approximately the same tidal level to minimize the difference in growth 7 page 3 conditions related to differential exposure to the water. The mussels for each area were kept in separate aerated aquariums and supplied with a continual flow of sea water at ambient temperature. They were allowed several days to adjust to the new situation and to begin normal feeding activity (Macginitie 1941). The organisms used in the experiments were divided into three size groups. Size I organisms were 0.8-1.2 cm. in length, size II organisms 2.8-3.2 cm., and size III organisms 4.8-5.2 cm. Measurement was made at the widest point of the shell parallel to the ventral edge. These young organisms were chosen for the experiments to decrease the probability of spawning during the experiment, thereby minimizing the possibility of a large change in lipid content and possibly also DDT content during the experiments. Though M. californianus has been observed to be sexually mature at a length of 25 mm. and an age of approximately 4 months, it does not appear to spawn until the age of about 1 year (Coe and Fox 1942) at a length of approxi¬ mately 80 mm. (Fox and Coe 1943). Young animals were also chosen for the experiments because of their ability to adjust more quickly to experimental situations (Jørgensen 1960, 1966; Macginitie 1941; Coe and Fox 1942). DDE levels in natural populations were measured directly by gas chromatography. Samples were extracted and prepared for chromatographic analysis according to the method described by Stanley and LeFavoure (1965). Analysis of the extracts was page 4 performed on a Beckman GC-4 equipped with an electron capture detector. The chromatograms were performed isothermally (20090) on 3 per cent QF-1 on Chromosorb W, 80-100 mesh treated with DMCS. The carrier gas was helium. All of the experiments involving C uptake were run in a similar manner. Prior to incubation of the mussels, the shells were scraped free of barnacles, limpets, algae and other attached organisms. The animals were then placed vertically on a flat surface on absorbent paper to allow water in the mantle cavity to drain out. This not only induced the mussels to take in water soon after placement in the DDT solution (Fox, Sverdrup. and Cunningham 1937), but it also helped clear any food or inorganic particles suspended in the mantle cavity water. All experimental organisms were incubated in closed jars to minimize the loss of DDT from solution through codistillation (Acree, Beroza, Bowman 1963). Since previous studies have shown that Mytilus can filter particles as small as 1 u (Jørgensen 1960; Jorgensen and Goldberg 1953) and oxyhemoglobin particles as small as 40-50 A (Fox, Oppenheimer, and Kittredge 1953), the sea water to be used in the containers was millipore filtered successively through 8 u, 0.45 u, and 0.22 u pore filters to insure that no particles greater than 0.22 u remained in solution. In early experiments one organism was placed in each con- tainer. Size I organisms were incubated in 60 ml. water in a half-pint jar, size II organisms in 180 ml. in a one-pint jar, and size III organisms in 540 ml. in a one-quart jar. In later 2 page 5 experiments three organisms, one of each size class, were placed in each one-quart jar with 780 ml. filtered sea water. Animals were incubated in concentrations of 10 p.p.t., 100 p.p.t., 1.0 p.p.b., and 1.4 p.p.b. C -DDT for 12 hours. Some of the animals were assayed for uptake immediately at the end of the 12 hours, while others were kept 23 hours in normal sea water before being tested to show the level of DDI retained. The amount of C14 uptake was determined by dissecting the animal, taking its wet weight, homogenizing it in 1,4-dioxane, and counting in the scintillation counter. Size I organisms were homogenized twice in 14 ml. dioxane, size II organisms twice in 3 ml. dioxane, and size III organisms twice in 6 ml. dioxane. The extracts from the two homogenates were combined, and a 2 ml. aliquot was placed in toluene solution and counted 2 minutes in a Nuclear-Chicago Unilux II scintillation counter. The primary foods of Mytilus are diatoms, dinoflagellates, and detritus (Field 1921-1922; Buley 1936; Macginitie 1941; Coe and Fox 1942). The diatom Nitzschia closterium minutissima was chosen for the feeding experiments because it was readily filtered out of suspension and digested by Mytilus (Schultis 1969). C+-DDT uptake from phytoplankton was tested in a similar manner to earlier uptake experiments. A sample of Nitzschia consisting of approximately 1.24 x 10 cells was taken from a pure culture and incubated in 6.6 p.p.b. C-DDT for 64 hours at room temperature in diffuse light. This concentration of C page 6 DDT does not appear to harm the phytoplankton (Bailey 1969). The bottles were periodically shaken to keep the phytoplankton in a uniform suspension, and at the end of the incubation period, the solution was centrifuged and rinsed twice with filtered sea water to remove any DDT which might have adsorbed to the surface of the phytoplankton. The phytoplankton were then introduced to four experimental jars with 3 organisms each, one from each size class. About 2.5 x 10' cells were provided per container. Both the experimental conditions in the jars and the treatment of the animals at the end of the 12-hour incubation period were identical to those in the experiments with C uptake from water alone. ESULTS Assays with the gas chromatograph revealed a distinct difference in DDE levels in organisms of different sizes but a minimal difference between organisms from different areas (Table 1, Figure 1). Total DDE content was seen consistently to decrease with increasing size of the organisms. The concen¬ tration of DDE is expressed in terms of wet weight of all the body tissues combined. Table II shows the correlation between wet and dry weight for the three size classes. However, the relationship between size class and DDE level appears to be identical whether plotted against wet weight or dry weight. The experiments with the uptake of C+-DDT directly from the water revealed a similar trend of DDT accumulation with size. O page 7 The preliminary experiments showed that the size I organisms (0.8-1.2 cm.) concentrated DDT 20 to 70 times greater than did the size III organisms (4.8-5.2 cm.). This level did not significantly decrease 23 hours after termination of the incu¬ bations which suggests retention of virtually all DDT taken up during the experiment. However, correction of these values was necessary to account for the small organisms having available a greater volume of water per unit body weight than the large ones. The correction factor reduced the relative uptake of size I organisms to approximately 5 tines the uptake by size III organisms (Table 3). For further verification of this prediction, three organisms, one of each size class, were tested in the same container such that each would initially have available the same total amount of water. Five replicates of the experi- ment were made and the initial level of C-DDT in the water was the same for each container, 1 p.p.b. After incubation for 12 hours, the animals were removed and assayed. The results are shown in Table 4 and Figure 2. The same trends noted earlier were found, and the earlier corrected values were found to be valid, as size I organisms concentrated C+-DDT approximately 5 times more than size III organisms. This same tendency was again evident in the experiments with the uptake of C-DDT from Nitzschia (Table 5, Figure 3). The smaller Mytilus concentrated the DDT 4 to 4 times greater than did the larger ones. However, note that while the same trend is apparent, the total uptake was far less than the uptake 75 O 0 page 8 directly from the water. Furthermore, it is distinctly possible that this small degree of C+ uptake from a potential food source was a result of both uptake from the food itself and from C which had been released into the water by the labeled Nitzschia. DISCUSSION There are several possible explanations for the variation in DDE accumulation between large and small Mytilus. This trend could easily be understood if the lipid content of the mussels showed a similar decrease with size, but instead an increase occurs. Much of the lipid in a mussel is stored in its gonads (Rodegker and Nevenzel 1964), and as the animal grows, this organ greatly increases in size, extending into the mantle and surrounding many of the tissues. Shortly before the mussel spawns, the dry weight of the gonads may be equal to or greater than that of all the other body tissues combined (Coe and Fox 1942). Fat is deposited in the connective tissues of the gonads, causing a direct relationship between total fat content and the size of the reproductive organs. In these experiments it was noted that the gonads of the larger organisms were very exten¬ sive, and there was no evidence that the animals had recently spawned thereby lowering their lipid content. One would predict from the increase in lipid content of larger mussels that they would take up more C-DDT than the smaller ones. However, this was not the case. 7 C O page 9 The relative level of DDE could be higher in the smaller organisms if only the larger ones had the capability of metabo- lizing and excreting the pesticide. However, other studies have shown no such breakdown occurring in either small or large M. californianus (Davis and Comrey 1969). The findings of this study may be related to the fact that young Mytilus grow, filter, and adjust more rapidly than older ones when disturbed (Coe and Fox 1942). The average amount of water filtered by M. californianus decreases with age (Fox, Sverdrup, and Cunningham 1937) and as a possible consequence, the amount of DDT taken up from the environment via both the water and phytoplankton may decrease. Mussels may also cease filtering when disturbed. Jørgensen (1960, 1966) was able to obtain normal filtering rates in the lab only from very small mussels. Furthermore, though a mussel normally feeds 97-99%, of the time (Loosanoff 1942), it may stop eating when disrupted, the younger ones adjusting more quickly to experimental situations. In addition, a mussel may transport water without feeding if the sheet of mucus normally covering the gills is not present (Macginitie 1941). As a result, a mussel could take up more DDT directly from the water than through feeding on phytoplankton. The results of this study show that substantial quantities of DDT are obtained directly from the sea water. Furthermore, they suggest that this mode of entrance may be even more impor¬ tant in the uptake of DDT than the accumulation from feeding on contaminated food materials. However, more information is O page 10 needed to fully evaluate these findings. If indeed Mytilus is typical of marine organisms in its ability to take up so much insecticide directly from the sea water (Kaplan 1969; King 1969; Phillips 1969; Sutton 1969), then the marine organisms could be in even greater danger from DDT pollution than originally thought. 78 C FIGURE LEGENDS Figure 1. The solid colored bars represent organisms from Seal Rock, the horizontally crossed bar an organism from Point Joe, and the cross-hatched bar an organism from Cabarillo Point. The first three bars show mean DDE levels for 50, 10. and 6 mussels, respectively, while the last three show levels for individual animals. Figure 2. Each graph shows the uptake over a 12-hour period of C-DDT directly from the water by a l cm., a 3 cm.. and a 5 cm. Mytilus incubated together in the same jar. The initial C+-DDT concentration was 1.0 p.p.b. in 780 ml. filtered sea water. Figure 3. Each line represents the uptake over a 12-hour period of C-DDT from Nitzschia by a l cm., a 3 cm., and a 5 cm. Mytilus incubated together in 780 ml. filtered sea water. + 3 O + + + N e 90 N R + t ++ + + + +++ + ++ + + + ++ + + ++ +++ ++++++ + + t + ++ ++ + ++ P P p + + + + ttt t t ++ P ++ + + P + + +++ + + + O P ++ O + ++++ + + +++ 1 0 O + + + + + ++++ + H + + lt O + + + + ++ +++ + + ++ + +++ ++ 12- 10- pp.b D6 1000 2000 3000 4000 5000 H 600 400 200- 600 400 ».pb. DD 200- 600 400 200 Jar 1 Jar 2 Jar 4 1000 2000 3000 4000 5000 Jar 3 Jar 5 1000 2000 3000 4000 5000 C SUMMARY 1. In natural populations Mytilus californianus of lengths 1, 3, and 5 cm. have been found to have concentrations of DDE of 120, 96, and 62 p.p.b., respectively, per wet weight of body tissues. 2. Mussels can take up substantial amounts of DDT directly from the surrounding sea water. 3. Over a 12 hour period mussels 1 cm. in length take up 5 times as much C+-DDT directly from the water as animals 5 cm. in length. 4. The uptake of C+-DDT from phytoplankton by the smaller mussels is 4-44 times greater than the uptake by the larger animals over the same time period. 5. The amount of water filtered by a mussel decreases with age and may account for the more rapid uptake of DDT by younger organisms. 6. The ability of younger mussels to adjust to experimental conditions more readily than older ones may also partially explain the differential uptake rates. 7. It is suggested that if Mytilus is typical of marine organisms in its ability to concentrate DDT directly from the surrounding sea water, then the marine pesticide pollution problem may be greater than originally thought. REFERENCES Acree, F., M. Beroza, and M. C. Bowman. 1963. Codistillation of DDT With Water. Agricultural and Food Chemistry, 11: 278-280. Bailey, S. 1969. Personal Communication. Buley, H. M. 1936. Consumption of Diatoms and Dinoflagellates by the Mussel. Bulletin. Scripps Institution of Ocean- ography. Technical Series, 4:19-27. Coe, W. R., and D. L. Fox. 1942. Biology of the California Sea-Mussel (Mytilus californianus) I. Influence of Temp- erature, Food Supply, Sex and Age on the Rate of Growth. Journal of Experimental Zoology, 90:1-30. Davis, J. D., and C. Comrey. 1969. Personal Communication. Field, I. A. 1921-22. Biology and Economic Value of the Sea Mussel, Mytilus edulis. Bulletin of the United States Bureau of Fisheries, 38:125-259. Fox, D. L., and W. R. 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Comparative Biochemistry and Physiology, 11:53-60. Schultis, S. 1969. Personal Communication. Stanley, R. L., and H. T. LeFavoure. 1965. Rapid Digestion and Cleanup of Animal Tissues for Pesticide Residue Analysis. Journal of the Association of Official Agricultural Chemists, 18:666-667. Sutton, J. 1969. Personal Communication. 86 C Range of Length (mm.) 8.0-12.0 29.0-31.0 19.0-50.9 - - Mean Length (mm.) 9.1 29.9 19.7 19.3 50.5 52.0 TABLE 1. DDE LEVELS IN MYTILUS Mean Collection Wet Wgt. Indi¬ Site viduals (mgm.) 50 Seal Rock 22.5 851 10 Seal Rock Seal Rock 3580 3780 Point Joe 1150 Seal Rock 4810 Cabarillo Point p.p.b. DDE (per wet wgt.) 120 96 62 16 31 C 9 C Length (mm.) 8.2 8.9 10.5 10.5 11.0 28.8 29.2 9.8 31.4 31.5 49.1 50.5 51.0 51.0 51. TABLE 2. WET WEIGHT VS. DRY WEIGHI - Wet Wgt. Mean Dry Dry Wgt. Wgt. (mgm.) (mgm.) (mgm.) 6.9 3.3 12.4 4.9 1.5 7.9 28.0 27. 10.1 6.9 719 116 839 187 235 176 255 191 1072 165 625 3607 609 3906 894 5232 4413 750 2808 109 651 — O C 0 % + 8 - 5 8 a pO + . 0 NO 1 1 + 1 o O 1 aataava- 1 8 - O oo O( p op .O- 18.0 TABI 2.C BY 3 1Z. S O • (mgm. 18.8 OM WI Upta ke (p.p.b. — " e C TABLE 5. CH-DDT UPTAKE FROM NITZSCHIA S OF MYTILUS BY 3 SIZ — -DDT Mean C Size Mean Wet Range (mm.) Wgt. (mgm.) uptake (p.p.b.) 22. 8.0-12.0 8.70 28.0-32.0 1010 3.03 3957 18.0-52.0 2.01