2. CHROMIUM CONCENTRATIONS IN TUNICATES (UROCHORDATA: ASCIDIACEA) TAKEN NEAR MONTEREY, CALIFORNIA; RELATION TO LOCAL SEWAGE OUTFALLS HENRY HOWARD STIBBS Hopkins Marine Station of Stanford University, California Ascidians are known to concentrate such heavy metals as titanium, vanadium, niobium, and chromium in levels that tremen- dously exceed the concentrations of these elements in the surround- ing seawater (Webb, 1939; Bertrand, 1950; Carlisle, 1958; Le- vine, 1962). Chromium is a common constituent of industrial sewage, and it may at times constitute part of domestic sewage. Monterey, Bay, California, has a number of both industrial and domestic sewage outfalls. Using atomic absorption spectropho- tometry, a study was made of the chromium content of ascidians living in the Monterey area to determine the chromium levels present in different species and to see whether intraspecific variations in chromium content might be correlated with the lo- cations of selected sewage outfalls. MATERIALS and METHODS Eighteen species of ascidians were taken from the follow- ing four locations near Monterey: the floats at the marina in Monterey harbor; the rocky shore by the Hopkins Marine Station in Pacific Grove; the rocky shore at Point Pinos, Pacific Grove, 50 to 100 feet from the opening of the city sewage outfall; and the rocky shore south of the mouth of the Carmel River, 2 500 to 1000 feet from the opening of the city of Carmel sewage outfall. No ascidians were found living less than 50 feet from the Pacific Grove outfall and 500 feet from the Carmel outfall, although the rocks nearer to the outfalls appeared, but for the sewage, very suitable for ascidian habitation. Living solitary and colonial ascidians were picked clean of gross extraneous material and placed in finger bowls filled with millipore-filtered seawater to allow the gut to clear. The water was changed 2-3 times daily and the bowls maintained at about 14°0. The intestines of the ascidians were usually empty of feces within 48 hours, but some of the smaller ascidians (e.g., Clavelina huntsmani) closed their siphons in filtered seawater and never thoroughly evacuated. The large solitary ascidians (e.g., Styela, Ciona, and Ascidia) gener- ally proved much hardier than the smaller colonial species. After the ascidians were removed from the filtered seawater, all external adhering material was picked and scraped from the tunics as thoroughly as possible. An approximate wet weight was obtained, then the ascidians were either chopped up by hand or ground for several minutes in a Waring blendor. The chopped or ground samples were placed in 50 ml beakers and dried for 12-18 hr at 110°C to constant weight. A dry weight (to the nearest mg) was obtained for each sample. Samples were then ashed in a muffle furnace at 450°C for 8-12 hrs (24 hrs at 475°C for some samples, see Table 1) and weighed again. Aliquots of the ashed samples (usually O.5 g) were taken, weighed (to the nearest mg) in 15 ml beakers, and 3 ml of aqua 261 regia were added to each beaker. Samples were heated to dryness over a steam bath. Next, 2-3 ml of cold, concen- trated (30%) hydrogen peroxide were added to each beaker, and the samples were again completely evaporated over a steam bath. Finally, 3 ml of aqua regia were added to each sample and this evaporated as before. All samples contained distinct crystals of digested material at this time. Samples were redissolved in 10 ml of O.1 N HCl (prepared with deionized distilled water). This solution was poured into a 50 ml beaker. An additional 10 ml of the O.1 N HCI was used to rinse the 15 ml beaker, and this was also added to the 50 ml beaker. This 20 ml of solution, which often contained considerable undigested material, was filtered through 5 Whatman filter paper to remove macroscopic suspended mater- ial and larger pieces of sand and unidentified micaceous grit. An additional 5 ml of O.1 N HCl was used to rinse the 50 ml beaker and was filtered with the rest of the sample. The 25 ml of filtrate representing each sample was col- lected in a 50 ml Erlenmeyer flask and tested for chromium using a Perkin-Elmer Model 303 atomic absorption spectropho- tometer equipped with a three-slot burner head and an air- acetylene flame. The detection limit of this instrument is about 0.004 ppm chromium and the sensitivity (concentration needed to produce 1% absorption) was sometimes as great as 0.0713 ppm chromium. Standard solutions, containing 1, 5, 10, 15, and 20 ppm chromium, were made up using a Beckman stock solution of 1000 ppm chromium (prepared especially for atom- ic absorption procedures), diluted with the same O.1 N HCI solution that was used in making up the samples. Working curves, showing absorbance (derived from per cent absorp tion) plotted against ppm chromium, were used in determining concentrations of chromium in the samples. Appendix 1 shows one of the working curves obtained from the chromium standards. Fresh standard solutions were made up and tested prior to ana- lysing each group of ascidian samples. Since each 0.5 g ashed aliquot was dissolved in 25 ml of solution, the sample concen- trations read off the working curves were multiplied by 50 in order to obtain values in parts per million per gram of ash. Since glassware at the Hopkins Marine Station is cleaned with a solution of potassium dichromate and sulfuric acid, all of the beakers, flasks, and pipets used in these experi- ments were submerged in a solution of concentrated nitric and sulfuric acids for 15 minutes, then rinsed 6 times with tap- water, in order to prevent any chromium contamination. Control samples were prepared and tested for chromium to ascertain that no contamination was occurring at any stage of sample preparation. Tapwater, deionized water,aand the O.1 N HCl (the "blank") were all tested with the spectrophotometer and found to lack chromium. A 50 ml beaker was spared the usual washing with concentrated acid and filled with 25 ml of the 0.1 N HCl; this control, which might have shown contamination resulting from the washing procedure at the Hopkins Marine Station, also exhibited no chromium content when tested. One beaker was run through the gamut of sample preparation proce- dures and found to contain no chromium. Finally, the aqua re- 2d gia was tested and it, too, showed a lack of chromium. Neither the acid solution used in washing the glassware nor the concentrated hydrogen peroxide were run through the spectrophotometer, however. Samples of sewage effluent were obtained from local sewage treatment plants (Monterey, Pacific Grove, and Car- mel) and analysed for chromium using the method of additions. In this procedure, matrix interference is obviated by adding increasing amounts of chromium to a series of sewage samples; the atomic absorption readings are plotted against the chromium additions (Figures 1-4), and the line connecting the points extrapolated to the abscissa. The distance of this intersection to the left of the zero point of additions on the abscissa represents the chromium concentration in the unadulterated sample. RESULTS and DISCUSSION Monterey sewage sampled on May 29, 1970, contained approximately 0.008 ppm of chromium (Figure 1). No chromium was detected in the sewage effluents of Pacific Grove and Carmel (Figures 2 and 3). Seawater taken from the east shore of the Hopkins Marine Station in Pacific Grove contained 0.095 ppm of chromium (Figure 4). The open ocean contains about 0.00005 ppm of chromium (Goldberg, 1963). Chromium levels found in the eighteen species of ascidians tested are tabulated in Appendix 2 and summarized in Table 1. The ascidians tested contained chromium levels (ppm dry weight) 2 015 .010 .005 O.00 FIGURE Chromium Content of Monterey Sewage .008 CI Added ppm 20 01 .010 9 005 0.00 FIGURE 2 Chromium Content of Pacific Grove Sewage CI Added ppm 6 .015 o.010 .005 0.( FIGURE Chromium Content of Carmel Sewage CI Added ppm 20 2 .01 o.010 005 0.00 095 FIGURE 4 Chromium Content of Mussel Point Seawater C I Added p p m .20 70 at least 31,800 times, and in some species as much as 3,050,000 times, that in seawater in the open ocean. Species of Synoicum, Ritterella, and Eudistoma were found to contain the highest levels of chromium. These genera are all members of the Order Aplousobranchia; this fact is of particular interest, since the ascidians showing high levels of vanadium fall mainly in the Order Phlebobranchia (Webb, 1939; Bertrand, 1950). Within Orders and Families of ascidians there is consid- erable variation in the tendency to concentrate chromium. How- ever, all samples of Eudistoma species contained relatively high concentrations of chromium, whereas samples of Amaroucium species were consistently lower. The levels found in Eudistoma ritteri (zooids plus tunic) are somewhat lower than those ob- tained by Levine (1961), who found 72-144 ppm (dry weight) for whole zooids, and 100 ppm for zooid abdomens only. It is apparent (Table 1) that chromium levels in ascidians taken near sewage outfalls were generally lower than those in ascidians collected in an area considered unpolluted (Mussel Point, Hopkins Marine Station). In the case of the ascidians taken from the region of the Carmel outfall (indicated by an asterisk in Table 1), it is possible that the low levels reflect a difference in procedure. These animals were prepared in the same general way as the others, but were ashed for 24 hours at 475°C instead of the usual 8-12 hours at 450°0, and the low chromium values here may be due to vaporization of some chromium-containing component of the ash. Since these values appear abnormally low, they are not considered further here. Ascidians living near the Pacific Grove sewage outfall at 7 TABLE 1 Chromium content of Ascidians; Mean and Range, ppm in Ash Sampling Monterey Point Hopkins Location Marina Marine Pinos Station Species Order APLOUSOBRANCHIA Family SYNOICIDAE 4.0* Amaroucium californicum 14 12.75 Amaroucium solidum (11.5-14) 12 8 Polyclinum planum 105 98.7 Synoicum par-fustus (97.4-100) 10* 85 Ritterella pulchra 61 Ritterella rubra Family POLYCITORIDAE 6.75 21.5 Clavelina huntsmani (6.5-7.0) Cystodytes lobatus 9. 8.5 Distaplia occidentalis (9.0-10.5 Distaplia smithii 18.66 Eudistoma molle (17-19.5) 110 162.5 Eudistoma psammion (150-170) 25.75 30.25 Eudistoma ritteri 3-32.5) (24.5-27) Carmel Sewage Outfall 10.75 (10.5-11) 6* 5.5* 10* 7.25* 7.5* 11.5 6.75* 88.75 (86.5-91) 14.25* 27 TABLE 1 (continued) Hopkins Sampling Monterey Point Location Marine Pinos Marina Station Species Order PHLEBOBRANCHIA Family ASCIDIIDAE Ascidia ceratodes 9.5-12.5 Family CIONIDAE 6.75 Ciona intestinalis (6.5-7.0 Family PEROPHORIDAE Perophora annectens Order STOLIDOBRANCHIA Family BOTRYLLIDAE 12.8 Botrylloides sp. 9.5-17.0 Family STYELIDAE 4. Styela montereyensis (4-5) * Denotes samples that were ashed for 24 hours at 475°C inst the usual 8-12 hours at 450°C. Carmel Sewage Outfall 12.58* 6.1* ad o 7 7 Point Pinos generally tend to contain less chromium than do those on Mussel Point by the Hopkins Marine Station. It is possible that this tendency is due to the higher concentration of chromium in the seawater off Mussel Point. Water here, however, was not compared to seawater in other localities. It is also possible that Pacific Grove sewage effluent has some influence on the ability of Point Pinos ascidians to concentrate chromium. Al- though it has been shown to lack detectable amounts of this heavy metal, Pacific Grove sewage effluent flows into the ocean as fresh water 1-2°C warmer than the seawater at Point Pinos. Furthermore, in the period of the present study this sewage at the outfall itself was found to contain residual chlorine levels up to 45 ppm. Perhaps one or more of the components of this sewage lowers the chromium-concentrating capacity of the Point Pinos ascidians. Another possibility is that the plankton con- sumed by the ascidians is being affected by the sewage. While the exact mechanism by which ascidians concentrate heavy metals is still debatable, it has been suggested that since chromium exists in such low concentrations in the open ocean, ascidians may acquire this metal in a preconcentrated form in their plankton meals (Levine, 1962). SUMMARY 1. Eighteen species of ascidians taken near Monterey, Cal- ifornia, were analysed for chromium content using atomic absorp- tion spectrophotmetry. 2. The highest values (ppm dry weight) were found in the genera Eudistoma (E. psammion, 152.5; E. ritteri, 25.6; E. molle, 15.0), Synoicum, (Synoicum par-fustus, 83.6), and Ritterella (R. pulchra, 69.6; R. rubra, 45.2). All are mem- bers of the Order Aplousobranchia. The lowest value was ob- tained in Styela montereyensis (1.59 ppm dry weight). Seawater taken by the Hopkins Marine Station yielded a value of 0.095 ppm. 3. The chromium content of ascidians found near local domestic sewage outfalls was often lower than that of ascidians found in less polluted areas. The chromium concentration in three different samples of domestic sewage effluent (ranging from 0.000 to 0.008 ppm) was much lower than that in seawater from Monterey Bay.(0.095 ppm). LITERATURE CITED Bertrand, D., 1950. Survey of Contemporary Knowledge of Biochemistry, 12: The Biochemistry of Vanadium. Bul- letin of the American Museum of Natural History. Vol. 29: Article 7, New York. Carlisle, D. B., 1958. "Niobium in Ascidians," Nature, 181:933. Goldberg, E. D., 1963. "The Oceans as a Chemical System, p. 3-25. In M. N. Hill (ed.), The Sea, Vol. 2. Inter- science. Levine, E. P., 1962. "Studies on the Structure, Reproduc- tion, Development, and Accumulation of Metals in the Colonial Ascidian Eudistoma ritteri Van Name, 1945, Journal of Morphology, Vol. 111, No. 2. D. A., 1939. "Observations on the blood of certain Webb, ascidians, with special reference to the biochemistry of vanadium," Journal of Experimental Biology, 16:499- 523. FOOTNOTES 1. This work was supported in part by the National Science Foundation Undergraduate Research Program, Grant +GY-7288. The author wishes to express his gratitude to Dr. Donald P. Abbott, under whose guidance this work was carried out; and to acknowledge with appreciation his gen- erous help with identification of species and arrangement and discussion of data. 1.0 0.9 0.8 0.7 Ouos 0.5 8 o 0.4 0.3 0.2 0.1 0.0 20 CHROMLUM A PPEN WORKING URN 15 RO MI UM 7 Species Location Weight Dry Weight Dry Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. Amaroucium califor¬ nicum Hopkins 11.9g 1.083g 1.083 .551g .5g 4 2.03 .184 APPE NDIX 2 ASCIDIAN SAMPLES Amaroucium Amaroucium califor¬ califor¬ nicum nicum Carmel Point Outfall Pinos 18g 25.2g 2.158g 1.648g 2.Og 1.648g 1.464g .775g 5g 5g 10.5 14 7.7 6.6 .66 .605 Amaroucium califor¬ nicum Carmel Outfall 24.7g 2.159g 2.Og 1.445g .5g 11 7.95 .695 Amaroucium solidum Hopkins 43.3g 3.75g 2.Og 1.591g .5g 14 7.96 6.87 279 Species Location Wet Weight Dry Weight Dry Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. Synoicum par-fustus Hopkins 42.9g 2.740g 2.Og 1.674g .5g 100 83.6 5.35 SAMPLES Synoicum Synoicum par-fustus par-fustus Point Hopkins Pinos 43.5g 6.1g 2.762g 1.086g 2.Og 1.086g 1.691g .847g .5g .5g 97.4 105 82.3 82 5.23 14.6 Synoicum par-fustus Carmel Outfall 40.8g 7.207g 7.207g 5.768g 5g 5.5 4.4 .78 Ritterella pulchra Hopkins 2.8g .311g .311g .206g .201g 10 6.62 .735 Species Location Wet Weight Dry Weight Dry Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. Ritterella pulchra Point Pinos 36.2g 4.038g 4.038g 3.306g .5g 85 69.6 7.8 SAMPLES Ritterella Ritterella rubra rubra Carmel Point Outfall Pinos 15.7g 12.1g 2.127g 1.635g 1.635g 2.127g 1.574g 1.081g .5g .5g 10 61 45.2 6.62 6.14 .895 Clavelina huntsmani Hopkins 25.8g 1.274g 1.274g .840g 5 21.5 14.2 .703 Clavelina huntsmani Hopkins 42.6g 2.105g 2.105g 1.617g 5g 21.5 16.5 1.3 Species Location Wet Weight Dry Weight Dry Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. Amaroucium solidum Hopkins 43.2g 3.143g 2.Og 1.55g .5g 11.5 8.91 6.49 SAMPLES Amaroucium Amaroucium solidum solidum Carmel Point Outfall Pinos 23.9g 59.6g 1.531g 3.835g 1.531g 3.835g 1.103g .862g .5g .5g 77 6 2.05 3.37 .216 .132 Polyclinum planum Hopkins 107.7g 3.437g 2.Og 1.176g 5g 12 7.05 .137 Polyclinum planum Point Pinos ——— 1.340g 1.340g .683g .5g 8 4.07g ———— Species Location Weight Dry Weight Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. Eudistoma molle Hopkins 51.6g 5.601g 2.Og 1.473g .5g 17 12.5 1.36 SAMPLES Eudistoma Eudistoma molle molle Hopkins Hopkins 53.1g 55.8g 5.806g 5.118g 2.Og 2.Og 1.491g 1.545g 5g .5g 19.5 19.5 14.5 15 1.58 1.37 Eudistoma ritteri Hopkins 53.0g 3.917g 2.Og 1.408g .5g 32.5 25.6 1.88 Eudistoma ritteri Hopkins 55.7g 4.007g 2.Og 1.462g 5g 28.0 20.5 1.48 Species Location Wet Weight Dry Weight Dry Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Or per gm. Wet Wt. Eudistoma psammion Hopkins 45.3g 10.533g 2.0g 1.783g .5g 167.5 149.5 34.8 SAMPLES Eudistoma psammion Hopkins 44.4g 12.066g 2.Og 1.808g .5g 150 135.5 36.8 Eudistoma psammion Hopkins 40.8g 8.500g 2.Og 1.791g .5g 170 152.5 31.7 Eudistoma psammion Point Pinos 25.Og 3.684g 3.684g 3.ooog 5g 110 74 10.9 Eudistoma psammion Carmel Outfall 39.6g 7.739g 2.Og 1.794g .5g 91 81.7 15.9 Species Location Wet Weight Dry Weight Dry Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. Distaplia occident¬ alis Hopkins 48.9g 3.4833 2.Og .699g .5g 9 3.14 .224 SAMPLES Distaplia Distaplia occident¬ ocident alis alis Point Hopkins Pinos 49.6g 62.2g 3.769g 5.427g 2.Og 5.427g .669g 3.761g .5g .5g 9 8.5 3.01 5.9 .514 .229 Distaplia occident¬ alis Carmel Outfall 39.6g 3.443g 2.Og .939g .5g 11.5 5.39 .468 Distaplia smithii Carmel Outfall 29.5g 2.072g 2.072g .858g .5g 6.75 2.8 .197 Species Location Wet Weight Dry Weight Dry Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. Clavelina huntsmani Point Pinos 49.2g 2.426g 2.426g 1.802g .5g 6.5 4.83 .238 SAMPLES Clavelina Clavelina huntsmani huntsmani Carmel Point Outfall Pinos 11.6g 52.2g .573g 2.575g .573g 2.575g 1.975g .314g .310g .5g 7.25 5.37 3.97 .196 .265 Cystodytes lobatus Carmel Outfall 25.7g 1.604g 1.604g .759g .5g 7.5 3.55 .222 Distaplia occident¬ alis Hopkins 49.5g 3.367g 2.Og .682g 5g 10.5 3.58 .243 Species Location Wet Weight Dry Weight Dry Aliquot Ashed eight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. Eudistoma ritteri Point Pinos 41.5g 3.Og 2.Og 1.184g .5g 27 16 1.15 SAMPLES Eudistoma Eudistoma ritteri ritteri Point Carmel Pinos Outfall 23.6g 45.1g 2.635g 3.5g 2.635g 2.Og 1.206g 1.723g .5g .5g 24.5 14.25 14.8 9.33 1.13 1.04 Ascidia ceratodes Monterey Marina 36.7g ——— 2.Og .871g .5g 12.5 5.94 ———— Ascidia ceratodes Monterey Marina 35.9g ——— 1.Og .469g .469g 9.5 4.46 ——— Species Location Wet Weight Dry Weight Dry Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. SAMPLES Ciona Ciona Ciona Ciona Perophora intestinalisintestinalis intestinalisintestinalis annectens (tunics (w/o tunics Monterey Monterey Monterey Monterey Carmel Marina Marina Marina Marina Outfall 20.3g 8.1g 53.9g 37.7g 47.2g 0.950g 2.032g 1.273g 1.675g 0.768g 2.0g 1.Og 1.Og 0.579g 0.871g 0.467g 0.270g 0.485g 0.5g 0.5g 0.267g 0.467g 0.485g 6.5 7.0 44.9 12.58 5.61 3.96 3.04 12.1 2.72 7.65 0.284 0.185 0.41 0.124 0.097 Species Location Wet Weight Dry Weight Dry Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. Botryl¬ loides sp. Monterey Marina 7.8g .547g .547g .210g .206g 17 6.55 .218 SAMPLES Botryl- Botryl- loides loides sp. sp. Monterey Monterey Marina Marina 53.3g 53.3g 4.020g 4.020g 1.Og 2.Og 1.184g 706g 5g .482g 9.5 11.9 8.4 5.62 .632 .423 Styela monterey- ensis Monterey Marina 42.9g 2.146g 2.Og .808g 5g 1.62 .81 Styela monterey- ensis Monterey Marina 48.1g 2.370g 2.Og .794g .5g 1.59 .0785 Species Location Wet Weight Dry Weight Dry Aliquot Ashed Weight of Dry Aliquot Ashed Aliquot Ppm Cr per gm. Ash Ppm Cr per gm. Dry Wt. Ppm Cr per gm. Wet Wt. Styela monterey- ensis Monterey Marina 48.5g 2.443g 2.Og .783g .5g 5 1.96 .095 SAMPLES Styela Styela monterey- monterey. ensis ensis Monterey Carmel Marina Outfall 17.3g 51.6g 1.978g 2.650g 1.978g 2.Og .514g .791g .5g .492g 5 6.1 1.98 1.59 .11 .182 29