ABSTRACT The area of Monterey Bay near the Monterey City Outfall Line was investigated for relationships between dissolved oxygen and nutrient levels and changes in biogeochemical cycles during a day- night study period. Consideration was given to the production of the toxins hydrogen sulfide and nitrite, the use of nutrients as indi- cators of sewage pollution in the marine environment, and correla- tions of nutrients and oxygen levels to time, depth, and proximity to the outfall. 2. It was found that neither oxygen levels decreased enough nor did hydrogen sulfide levels increase enough to pose immediate toxicity to fish life during the period of study. 3. Levels of hydrogen sulfide, onygen, and to a lesser extent, nitrite, are dependent upon a day-night cycle. Phosphate seems to be de- pendent to a greater extent upon the volume of effluent being released from the outfall. Nitrate shows no obvious correlation with either factor. Phosphate is found in greatest quantities at the surface. Hydrogen sulfide and nitrite are found in slightly increased levels towards the bottom depths. Nitrate does not show any obvious gradients with depth. Stability of phosphate seems to make it a better indicator of effluent flow direction and rate than the other nutrients studied. Using phosphate as an indicator, it is suggested that much of the effluent of the Monterey City Outfall is flowing southward and pool ing in a deadwater area near the Monterey Wharf No. 2, enriching the area considerably. 6. INTRODUCTION Virtually all the work so far reported concerning the effects-of sewage pollution on the marine environment has been based on studies made during daylight hours. There is little information concerning nu¬ trient and oxygen levels or fluctuations of biogeochemical cycles and environmental factors at night. This paper is devoted to an investiga- tion of the possible nighttime changes which might take place of oxygen and nutrient levels around a primary sewage outfall and to relate these to fluctuations in certain biogeochemical cycles as well as to depth, time, and proximity to the outfall. Of particular interest is the sulphur cycle which involves the breakdown of organic matter containing sulphur compounds (amino acids) to hydrogen sulfide, then to sulphur, and finally to sulfate, which can then be recycled by plants. This process is dependent upon the presence of aerobic conditions (fig. 1). If, however, the oxygen level is suf- ficiently lowered, then anaerobic bacteria such as Desulfavibrio can con¬ vert the sulfate directly to hydrogen sulfide, an extremely toxic sub- stance. Hydrogen sulfide occurs normally in bottom sediments where or- ganic matter is plentiful and anaerobic conditions exist. In times of upwelling and turbulence, when quantities of hydrogen sulfide are brought to the surface, massive fish kills can take place (Theede, 1968). Kor- mandy (1966) has made the same observations in areas polluted by the effluents of pulp mills. As the concentration of hydrogen sulfide in- creases, total biomass decreases proportionally. Theede (1968) further believes that organisms with greater mobility are much more sensitive to hydrogen sulfide poisoning than less mobile organisms, since the toxin e acts as an inhibitor of respiration. Thus, fish are usually more effect- ed by hydrogen sulfide than are most invertebrates. In aereated sewage systems, most hydrogen sulfide is broken down to sulphur because of the aerobic conditions. There exists, however, the possibility that around an outfall area substantial oxygen depletion could occur by aerobic bacteria breaking down accumulated organic matter in the effluent. This situation might become extreme during night hours when there is no photosynthesis to replenish the oxygen used up by the aerobic bacteria. This oxygen reduction might lead to anaerobic condi¬ tions sufficient to allow production of hydrogen sulfide in amounts cap¬ able of poisoning fish in the area. Processes occuring at night could then lead to toxin production that would go relatively unnoticed when sampling only during the daytime. In a similar fashion, nitrate can be anaerobically converted by denitrifying bacteria to the toxic nitrite phase (fig. 2). Nighttime conditions could also bring about changes in the nitrogen cycle that would not be noticed when samples were taken only during the day. This work will deal with relationships between oxygen and nutrient levels near the marine outfall of Monterey, California, with special consideration given to the production of both these toxins, hydrogen sulfide and nitrite, as related to changes in their respective biogeo¬ chemical cycles which could be caused by the presence of primary effluent. Finally, the validity of using nutrient levels as indicators of pollution and their relationships to time, depth, and proximity to the outfall will be considered. Figure 1. The Sulfur Cycle. Note the conver- sion of sulfate to hydrogen sulfide under an- aerobic conditions shown by heavy black line. Figure 2. The Nitrogen Cycle. Note the produc- tion of nitrite from ammonia under aerobic condi- tions and from nitrate under anaerobic conditions. 9. 2 sc sultate angerobic NO3 nitrate SULFUR CICIE R-SH prganie mailer angerobic sultur NIIROGEN CYCLE R- 62 organic matter NO2 nitrogen gas nitrite N20 nitrous oxide No2 nitrite hydrogen sultide NH. ammonia 15 METHODS AND MATERIALS The area studied is at the southern end of Monterey Bay, Californ- ia. It extends from the Monterey Commercial Wharf No. 2, approximately 3.5 miles northward to just south of the Fort Ord Firing Line (fig. 3). Within this area six stations were set up parallel to the long sandy Del Monte Beach Area, each station being about 800 feet offshore (fig. 4). The underlying bottom is mostly sandy, with a bed of shale occuring just south of station 3 and extending southward towards the wharf (Dorman, 1968). Between stations 3 and 6 there exists a large bed of the kelp Macrocystis integrifolia. The average depth of the stations is 30-40 feet for stations 1-5 and 20-25 feet for station 6. Station 1 is just south of the Fort Ord Firing Line offshore from a large sand and gravel company that is removing sediment from up to 100 feet offshore. Station 2 is opposite the outfall for the city of Seaside, about one mile south of station 1. Because the outfall line extends only 300 feet from shore, it was impossible to get close enough to take samples at the boil due to the heavy surf action frequently encountered in the area. Station 3 is at the boil of the Monterey City Outfall Pipe, one mile south of station 2. The outfall line extends 800 feet from the shore into Monterey Bay and releases about 217 million gallons per day of pri- mary treated sewage at a point 18 feet below the surface of the water. The boil can usually be detected from sea by the strong odor of chlorine, the water discoloration, and the relative smoothness of the water at the point where the effluent reaches the surface (fig. 5). 1 Stations 4 and 5 are located 400 and 800 yards, respectively, south of station 3. Station 6 is about 500 yards south of station 5 and 100 yards north of the Monterey Commercial Wharf No. 2. Beginning at station 3 and extending south to station 6 the water tends to be a bit more gcalm as the area becomes increasingly protected from wave action. Accordingly, on rough days when stations 1 and 2 could not be approached, stations 3-6 could be easily sampled. Water samples at three depths for each of the six stations were taken at a single time on each of eleven days. Sampling was scheduled to include two days at each of the following times: 0400, 0800, 1200, 1600, and 2400 hours. Only one series of samples was taken at 2000 hours. From aboard ship, water samples were taken by reversing Nansen bottles at the surface (0-2 feet), at mid (12-17 feet), and at bottom (20-30 feet) depths for analysis of dissolved oxygen and nutrients. Temp- erature and depth were both recorded by means of a Martek TDC Probe (Martek Instruments, Inc.). Figure 6 shows the sample stations, fre- quency of sampling, and water temperatures for each depth at each station. Until 20 May, 1970, dissolved oxygen at the three depths was deter- mined by the Winkler Method (Strickland and Parsons, 1969). After this date, a Martek Oxygen Probe was used. Oxygen samples for Winkler analysis were fixed on board ship in 300 ml glass BOD bottles and anal- yzed within 24 hours in the laboratory. Water samples were also analyzed for phosphate, nitrate, nitrite, and hydrogen sulfide. Nutrient samples were taken in 250 ml plastic bottles and frozen 24-48 hours. Analysis began after a thawing period of one to two hours. All nutrient analyses were done according to the methods outlined in Strickland and Parsons (1969), 128 Figure 3. Monterey Bay. The area of study is located in the southern end of Monterey Bay near the cities of Monterey and Seaside. miles MONTEREY one BAY Santa Cruz Monterey Canyon Moss (Landing AArea o Stud, Seaside 127 Figure 4. The Area of Study. Six stations were set up in the 3.5 mile area that runs from the Monterey Commercial Wharf No. 2 north to the Fort Ord Firing Line. The stations were sampled 800 from shore. Note the Monterey City Outfall Line which extends offshore 800 feet and the Saaside City Outfall Line offshore about 300 feet. Wind was always from the West. 952 05 0 2. a 5 a Z 8 Figure 5. The Monterey Outfall Pipe Boil. The boil is easily identified from ship by the strong odor of chlorine and water discoloration. Note the relative smoothness of the water (diametert8 m.) at the point where the effluent reaches the surface. (Photo by W. Cooke from aboard Hopkins Marine Station Research Vessel Tage. 19 May 1970. 38 Figure 6. Sampling Chart. Stations and depths sampled are shown in the left hand column. Dates of sampling at a certain time are shown above. Temperature of station and depth is shown for any date and time. When station was not sampled, (-) appears. When station was sampled, but no temperature was recorded an(*) appears. Note that temperature readings around the boil are not significantly different from other stations. O T — ION 2 (VI OE o — — —— — 0- O o 9* ** * 0 4 * o + ad ro . O- O . * 0. 1—— 00 O -— oq a — — o (V - -— - —— — — 10 - o 10 V - B 3. 3 3 so S8 SO aaa 0 RESUET Oxygen Throughout the 24 hour period sampled, levels of oxygen appeared to show similar trends at all six stations (fig. 7). In each case, ox- ygen reductions occurred just before sunrise (0400-0800). Oxygen levels then increased during the daylight hours until around 1600, at which point they begin to drop off again. They continued to drop throughout the night until midnight. The low point at midnight was then followed by an increase with a high point at 0400. Note that differences in dissolved oxygen between surface and bottom samples were not terribly large, although the bottom samples at all stations were more oxygen-depleted from 1600 to midnight. It is significant that the degree of oxygen variation (fig. 8) for a 24 hour period at station 3 is not decidely different from variations of ogygen levels at any other station during the same period. However, the lowest reading for oxygen of the entire survey (4.7 ppm) was observed at 2400 on the bottom of station 3. Only twice did dissolved ogygen levels at the surface drop below 6 ppm, and while bottom levels dropped below 6 ppm seven times, only once did they drop below 5 ppm. At no time did any station, at any depth sampled, have less than 50% oxygen saturation of the water. ydrogen Sulfide Varying amounts of hydrogen sulfide were found and for the most part appeared to bear a definite inverse relationship to levels of ox ygen at the depths sampled (fig. 9). The highest increases in hydrogen sulfide were observed from 040.080, which is the same time thet ovygen Figure 7. Oxygen Levels Over 24 Hour Period for Six Stations. Surface oxygenilevels are shown by broken line and bottom oxygen levels are shown by solid line. Note same basic trend for all six stations with the greatest oxygen depletion occuring at midnight on the bottom of all stations. High point at 0400 is believed to be due to water turbulence on days when sampling took place. Oxygen is shown in p.p.m. suriace 8 beie Sta.1 8 5ta. 2 8 Sta. 3 BOIL 0400 0800 1200 1600 2000 2400 TIME Sta. 4 9- Sta. 5 Sta. 6 Wharf 0400 0800 1200 1600 2000 2400 8 Figure 8. Means and Standard Deviations. Means are computed from all values of a parameter over 24 hour period. Deviations show variability of that parameter over same time period. Note in- creased variability around stations 3 and 6. Also note highest values around these stations. Nitrate, hydrogen sulfide,aand nitrite are all quite variable compared to the more stable (smal deviations) phosphate. Oxygen is measured in ppm, nitrate in ugm.-at. N/1., nitrite also in ugm.-at.N/1., phosphate in.ugm.-at. P/1., and hydrogen sulfide in ugm.-at. HoS/1. a 8 02 00 Surface 2 STATION Mid OXYGEN NITRATE PHOSPHATE NIIRITE HYDROGEN 5 L STATION 4 + Bottom STATION 5 Figure 9. Oxygen and Hydrogen Sulfide Levels. Oxygen is shown in p.p.m. and hydrogen sulfide in ugm.-at./1. Note inverse relationship observed between the two parameters. As one increases, the other decreases accordingly. Even at points of greatest hydrogen sulfide production, oxygen saturation never dropped below 60%. Station 5 values are from the surface. Stations 3, 6, and 1 show bottom values. 0 ee 0 UGM.-AL H8/1. levels showed their greatest decrease. As oxygen levels fell throughout the rest of the day, levels of hydrogen sulfide gradually increased. Note that even at the point of greatest hydrogen sulfide production (almost .3 ugm.-At. hydrogen sulfide/L. at station 6) oxygenllevels fell to no more than to 63% saturation. Once again, there does not appear to be a significant difference between samples taken at the different sta- tions, regardless of the depth sampled. Both surface and bottom samples show trends of hydrogen sulfide fluctuation throughout the day that are inversely proportional to those trends shown by oxygen levels. However, a slight increase in hydrogen sulfide production was observed towards the lower depths. Nutrients Neither phosphate nor nitrate showed any obvious correlations with oxygen levels as was observed with hydrogen sulfide. Nitrite, however, though not showing strict correlations during the day, did show increased concentrations near the bottom at station 3 and station 6, the two areas where oxygen depletion was greatest during the night. There also exists a rapid increase in nitrite levels at all stations from 0400-0800, again this being the period of large decreases in oxygen levels at all six stations (fig. 10). Computed means and standard deviations for concentrations of ni- trite, nitrate, and hydrogen sulfide indicate the study area as one that has highly variable nutrient levels (fig. 8). Again, note the presence of slightly increased concentrations of hydrogen sulfide and nitrite at the bottom depths. Also, phosphate concentrations are relatively stable compared to the extreme fluctuations shown by nitrate and hydrogen sulfide and, to a lesser degree, nitrite. 14 Figure 10. Nitrite Levels Over 24 Hour Period. Nitrite concentrations at surface depths are shown by solid black line and at bottom depths by broken line. Concentrations of nitrite are shown in ugm. at. N/1. Note the slightly increased levels of nitrite at bottom depths, especially at stations 3 and 6. Note also the rapid increase in all cases from 0400-0800. D 04 03 02 O1 04 +03 02 01 04 .03 02 0 Sta.1 suriace — bottom Sta. 2 Sta. 3 Boil 9— 0400 0800 1200 1600 2000 2400 8 9—0 0400 0800 1200 1600 2000 2400 Sta. 4 Sta. 5 Sta. 6 Wharf TIME 14 The highest concentrations of nitrite and phosphate, and some- times nitrate, are observed at station 3. However, the nutrient levels at station 6 are consistently high and the variability of each nutrient at station 6 is often equal to, and sometimes higher than, the variability of nutrients at station 3. It is noteworthy that the boil at station 3 seems to be the most variable of all six stations, especially in phos- phate and nitrate concentrations. Phosphates, which exhibit their greatest concentrations at the sur- face of station 3, tend to show lowered concentrations as they approach station 5. However, at station 6, though surface and mid depths still have fairly low phosphate levels, there exists a large increase in bottom phosphate concentrations, being almost as great as those found at the boil (fig. 11). There seems to be some correlation between volume of effluent be- ing released from the pipe and levels of phosphate found (fig. 12). After the effluent is brought up to maximum flow at 0800, phosphate rises markedly, and as the effluent flow decreases throughout the day, phos- phate levels gradually decrease. Phosphate levels are lowest during the dark, precisely the same time that the volume of effluent is being dis- charged at its minimum flow rate. This observation was made at station 3, but at station 6 this correlation does not hold. At station 6, while the highest phosphate levels were detected during the day, high readings (high- er than at the boil) were found during the dark hour also. Nitrate did not seem to bear any relationship to volume of flow as did phosphate, nor did nitrate show any increased concentrations at any specific depth. Phos- phate showed greatest levels at the surface of all stations. Figure 11. Phosphate Levels From Boil to Wharf. Phosphate is measured in ugm.-at. P/1. Circles and broken line are surface values. Triangles and dot-dash lines are mid depth values. Crosses and solid lines are bottom values. Note decreasing phosphate values as station 5 is approached, es- pecially in upper depths. Note also large bottom increase in phosphates at station 6. 14 9 ta ta- 114. 64) 14 Figure 12. Phosphate Levels, Nitrate Levels, and Volume of Effluent from Monterey Outfall. Flow of effluent is measured in millions of gallons/day. Phosphate and nitrate are measured in ugm.-at. P/1. and ugm.-at. N/1. respectively. Note increase of phosphate in morning, gradual decrease throughout day, and low concentrations at night. No apparent pattern is followed by nitrate. Surface phosphates (thick black line) seem to be greater than either mid (broken line) or bottom depth (thin black line) values. No correlations seem to exist for nitrate. 0400 0800 suriace botong 1200 1600 TIME 2000 Monterey Outfall Phosphate at Boil Nitrate at Boil 2400 47 CONCLUSTON Throughout the night hours a general drop in oxygen levels did occur at all stations, presumably due to the inability of plants and phytoplankton to photosynthesize and produce oxygen. The maximum level of dissolved oxygen was observed at 0400 at all stations and could be due to turbulence of the water on the specific days sampled. It has been reported that in lakes, the presence of waves, and whitecaps can increase the oxygen content of the water by 10 to 100 times, allowing increased absorption of oxygen from the air (Imhoff, 1966). Even on the bottom around the outfall the oxygen level rarely drop ped below 5 ppm, regardless of the day or time of day. Though most ma- rine fish can tolerate oxygen drops down to 3 ppm, they prefer at least a minimum of 5 ppm (Martin, 1968). However, it has been found that some species of marine fish can suffer up to a 25% decrease in optimum repro- duction when living in water at 4 ppm (Martin, 1968). Imhoff (1966) further reports that some states on the East Coast, including New York, recommend that dissolved oxygen levels never fall below 50% saturation for purposes of bathing, fishing, and shellfish culturing. At no time and at no station in our survey did the oxygen level drop below 50% saturation, and seldom did it drop below 65-70% saturation. It seems that even at the boil there does not exist any immediate danger to fish life in terms of dissolved oxygen content of the surrounding waters, at least within the time period studied. The evidence found indicates that levels of hydrogen sulfide are closely related to levels of dissolved oxygen. The portion of the sul- fur cycle which involves the anaerobic conversion of sulfate to hydrogen sulfide could indeed be taking place in the study area, yet the levels 16 of hydrogen sulfide produced are fairly low at all the stations sam- pled. The reason for the low levels of hydrogen sulfide could be 1.) high oxygen levels which prevent the anaerobic processes from taking place to any marked degree, 2.) chlorination of the sewage at the treat- ment plant which breaks down hydrogen sulfide in the following way: 8 t C1—HCl 8 and 3.) a scarcity of sulfur-containing compounds in the effluent itself It seems that most of the hydrogen sulfide is broken doun by the time it emerges from the pipe and that increases found were probably due primarily to anaerobic conversion of sulfate to hydrogen sulfide in the marine environment. These increases do not seem to be large enough, how- ever, to cause immediate poisoning to the marine fish in the area. But because this toxin was not found in great quantities in the water does not mean that hydrogen sulfide is absent from the area. For example, in 1951-1952, the Terminal Island Outfall in Los Angeles Harbor put out around 6 million gallons of primary treated sewage daily and oxygen levels were never reported below 4-5 ppm (Reisch, 1959). Though obvious quan- tities of hydrogen sulfide were not found in the water around the outfall Reisch believed that there were large amounts of sulfide in the sediments beneath the pipe, where conditions were more anaerobic. It is conceivable that in any period of extreme turbulence these toxin-containing sediments beneath the pipe could be stirred up, especially if the pipe is in rela- tively shallow water, and the poisons dispersed. It seems clear that dissolved oxygen levels are not always good indicators of what is happen- ing in the substrate in an area effected by an outfall. Sediments beneath Monterey Outfall could contain considerable quantities of hydrogen sulfide not necessarily detectable in the vater. 49 Throughout most of the day, nitrite concentrations increase probab- ly as a result of the aerobic breakdown of ammonia. But early in the morning after 0400, increases in nitrite could well be connected to the dropping oxygen levels which allow production of nitrite from nitrate under anaerobic conditions. Highest nitrite levels at the bottoms of stations 3 and 6 perhaps reflect the lower oxygen levels found at these stations, especially at night. While hydrogen sulfide seems closely correlated with oxygen levels, nitrite seems to be a bit less so, cor- responding inversely with oxygen only in the hours between 0400 and 0800. Nitrite seems to be produced under anaerobic conditions from nitrate and under aerobic conditions from ammonia whereas hydrogen sulfide levels found were probably all produced under anaerobic conditions from sulfate. The obvious relative stability of phosphate could mean that it may act as a good indicator of sewage pollution. Nitrate and hydrogen sulfide are too variable, and nitrite is not found in large enough quantities to act as an easily detectable indicator. Because of its close correlation with the volume of effluent leaving the pipe, phosphate may be used to possibly tell how much effluent is being dispersed and at what rate. Phos- phate seems to be more dependent upon effluent volume rather than on oxygen levels or any day-night cycle, as is hydrogen sulfide and, to a lesser degree, nitrite. If this is the case, then the phosphate gradient existing from sta- tion 3 to station 6 could indicate that the effluent is moving southward towards Nonterey Wharf No. 2 where it slows and particulate nutrients settle to the bottom as the effluent "pools" in this seemingly deadwater area. The high levels and high degrees of variability shown by all nu- trients at station 6 support this idea. Further evidence is given by Stevenson (1964) uho concluded that there exists water movement in the upper 15 feet from the boil forty-five degrees to the south a greater proportion of the time than in any other direction. There exists no seaward movement. Trumbauer (1966) found a slight increase in coliform bacteria towards the Wharf from the boil. Sherman (1970) believes that the Monterey effluent is the source of increased numbers of Proteus vulgaris which he found near the Wharf. I do not doubt that the fish companies and restaurants contribute to the large nutrient levels near the Wharf, but I cannot believe that these sources could raise nutrient concentrations to the levels noted in this stuch, Because nutrient concentrations at the Wharf approach those found within 50 yards of the boil and because the works cited indicated a southerly water movement from the Monterey Outfall, I believe that phosphate concentrations can be used as an indicator of effluent flow from the Monterey Outfall Pipe southward to the Monterey Wharf No. 2. There the effluent seems to pool and cause this area to be one of rela- tively high enrichment and pollution compared to other areas along Del Monte Beach. While it has been shown that neither oxygen decreases nor hydrogen sulfide increases reach levels sufficient to immediately poison fish, it is not known what effects the large increases in nutrients can have on organisms, especially as found at the Monterey Outfall and around the Monterey Wharf. Water movement south from the boil has caused an en- richment of water adjacent to the Wharf not much different from that seen around the outfall FERENCES Dorman, C. E. 1968. The Southern Monterey Bay Littoral Cell; A Pre- liminary Sediment Budget Study. USN Postgraduate School Thesis. 234 pp. Imhoff, K. and G. Fair. 1966. Sewage Treatment. John Wiley and Sons, Inc. 338 pp. Kormandy, E. 1969. Concepts of Ecology. Prentice-Hall, Inc. 209 pp. Martin, D. F. 1968. Narine Chemistry. Marcel Dekker, Inc. 280 pp. Reisch, Donald J. 1959. An Ecological Study of Pollution in Los Angeles- Long Beach Harbors, California. University of Southern California Press. 119 pp. Sherman, P. 1970. Personal Communication. Stevenson, 1964. Currents of Nonterey Bay. USN Post raduate School Thesis. 76 pp. trickland. J. and T. Parsons. 1969. A Practical Handbook of Sea- water Analysis. Fisheries Research Board of Canada. 311 pp. Theede, H. et al. 1969. Studies on the Resistance of Marine Bottom Invertebrates to Oxygen-deficiency and Hydrogen Sulfide, Marine Biology 2: 325-337. Trumbauer, D. S. 1966. Coliform Baceria Survey of Monterey Bay off Del Monte Beach. USN Postgraduate School Thesis. 150 pp. Turner, H. Ebert, E., and R. Given. 1966. The Merine Environment in the Vicinity of the Orange County Sanitation District's Ocean Outfall. Calif. Fish & Came 52: 28-48. ACKNOWLEDGEMENTS This work has been supported by the National Science Founda¬ tion Undergraduate Research Program Grant No. GY-7288. I wish to thank Dr. Welton Lee, my advisor, for his continuous enthusiasm, support, and encouragement in all aspects of this project. I am greatly indebted to Dr. Lee for his endless time and effort which he contributed to this work. Without the help of my fellow students who assisted me so many times in sample and data collection this paper would never have been completed. Finally, I want to express my personal thanks to Mr. Joe Balesteri who I am sure lost many hours of sleep in piloting our ship Tage at all hours of the night and day during the time of the survey. This paper is the result of the efforts of many people, some named here, and many others still unnamed. To all of them, I am grateful.