Environ. Sci. Technol. 1991, 25, 295-301
Isotopic Distribution of Carbon from Sewage Sludge and Eutrophication in the Sediments and Food Web of Estuarine Ecosystems Patrick J. Gearing,* Juanita N. Gearing,+ James T. Maughan,t and Candace A. Oviatt Graduate School of Oceanography, University of Rhode Island, Narragansett, Rhode Island 02882
Stable isotope ratios (8l3C) from samples of water, sediments, and biota traced the behavior of organic carbon for 3 summer months in estuarine mesocosms (three controls, three with added sewage sludge, three with added inorganic nutrients). Isotope ratios proved to be a useful quantitative tracer for sewage carbon as well as for the fresh phytoplanktonic carbon produced during nutrient fertilization. Sewage sludge sedimented within hours of its addition, and approximately 50% remained in sediments after 99 days. The sludge was not inert, but was biologically oxidized a t rates similar to those of phytoplankton carbon. Its residence time in the water column was too short for uptake by zooplankton, but it was readily assimilated by some benthic organisms. Fresh phytoplanktonic carbon from nutrient-induced blooms was isotopically heavy and thus distinguishable from old primary production (fixed before the experiment). It flowed through the pelagic and benthic food webs more extensively and more uniformly than did sludge carbon.
Introduction Large quantities of sewage sludge have been and continue to be discharged into the sea off many North American and European cities, yet its behavior and fate in the nearshore marine environment are not well understood. The many possible physical, chemical, and biological effects of sewage sludge (1-4) include the following: (a) heightened sedimentation, which could smother the benthic fauna; (b) eutrophication brought about by its high nutrient content; (c) poisoning of the biota by its toxic components; (d) organic enrichment, which can lead to population shifts and to anoxia due to increased respiration. The sediments are thought to be the final resting place of sewage sludge, but quantification of its accumulation rates has been difficult (5,6). Direct feeding studies on domestic animals show sewage sludge to be a relatively poor energy source (7, 8), yet is has often been hypothesized that opportunistic marine organisms use sludge as a food supplement in disposal areas (2). Quantitative data in support of this hypothesis are lacking. Stable isotope ratios of organic carbon make good qualitative tracers of sewage sludge because sludge, like other land-produced organic matter, is depleted in carbon-13 relative to that produced by phytoplankton (9). Four field-based studies have used this tracer. In the New York Bight, isotope ratios mapped the extent of sludge carbon dispersal and deposition in the sediments (10). In the Southern California Bight, Myers (6)was able to trace sewage sludge in the sediments; unfortunately, no mass balance could be calculated because measurements were unavailable of the quantity of sewage contributed but subsequently respired to COP In the same area, organisms (six samples of sole and three of prawns) collected near
* To whom correspondence should be addressed. Present address: 34, avenue des Retraites, Mont-Joli, Quebec, Canada, G5H 1E4. 'Present address: Maurice Lamontagne Institute, Fisheries and Oceans Canada, C.P. 1000, Mont-Joli, Quebec, Canada, G5H 324. *Presentaddress: Metcalfe & Eddy, Inc., P.O. Box 4043, Woburn, MA, 01888. 0013-936X/91/0925-0295$02.50/0
a major sewage outfall were depleted in carbon-13 relative to the same species (five and three samples, respectively) collected 130 km away, suggesting some assimilation of sludge carbon (11). A recent paper (12) showed similar trends for three species of fish and some benthic invertebrates. The methodological problem with such field studies is the lack of true controls with which to compare data from sewage-affected areas. It is also often difficult to obtain important information on the environment. The study reported here profited from the controls and the multiple physical, chemical, and biological data available from a major sewage sludge experiment a t the Marine Ecosystem Research Laboratory (MERL) (13,141 to quantify the movement of sludge carbon through the water column and into the sediments and food web of temperate estuarine mesocosms. A t the same time, phytoplanktonic carbon fixed during nutrient-fertilized eutrophic blooms was also followed. It has been previously noted that carbon produced during rapid growth of phytoplankton is isotopically heavy (15-18). The data provide a comparison of the behavior and biological availability of sludge carbon with carbon from primary production.
Experimental Section Experimental Design. Each MERL mesocosm is an outdoor cylindrical tank (1.83 m in diameter, 5.49 m high) containing 13000 L of flowing seawater (30-day residence time) and muddy sediments (0.37-m-deep tray) from Narragansett Bay. The water column was mixed and heated/cooled to mimic conditions in the adjacent Narragansett Bay. Both the water and sediments contain a natural assemblage of organisms. Details of the facility have been reported (19, 20). The experiment was conducted between June 14 and September 29, 1984 (99 days). Three mesocosms were untreated. Three received daily additions of fresh sewage sludge in a slurry with tank water (IS, 1.9 g of solids; 4S, 7.7 g of solids; 8S, 15.3 g of solids). Reagent grade inorganic nutrients (N, P, Si) were added to the final three tanks a t levels ( l N , 4N, 8N) roughly equalling the nutrient loading associated with the sludge. The rates of addition of sewage sludge are approximately the same as at the New York 106-mi site (lS),the New York 12-mi and Los Angeles Hyperion sites (4S),and the Seattle West Point site (8s).Additional descriptions of the sewage experiment can be found in Oviatt et al. (13, 14). Sampling. Combined, primary and secondary sewage sludge, digested anaerobically, was collected weekly from the Cranston, RI, activated sludge Waste Treatment Facility for addition to the mesocosms. Subsamples of the weekly batches were weighed, acidified (dilute HC1) to remove carbonates, washed, filtered (precombusted glass fiber filters), dried, homogenized and reweighed. Percent solids, percent organic carbon, and 613C were measured. Mesocosm sediments were collected with a corer specially designed to sample the unconsolidated, flocculent material a t the sediment-water interface (21-23). Samples were collected from each treatment before additions began, twice during the experiment, and at the conclusion. Each sample consisted of three cores from previously unsampled
0 1991 American Chemical Society
Environ. Sci. Technol., Vol. 25, No. 2, 1991 295
locations in the sediment. Cores were sectioned in 1-cm intervals (0-1,1-2, and 2-3 cm) and the three sections from each depth and treatment combined to provide a more representative sample of the total sediment. Sediments were treated in the same manner as sludge samples. Suspended particulates in water pumped from the top 1 ni of the water column during mixing were filtered onto precombusted glass fiber filters and air-dried before analysis. An aliquot of each water sample was allowed to settle and then examined microscopicallyto determine the types of phytoplankton or detritus present. Samples were collected every 2-3 weeks in the three control, 4S, 8S, and 8N tanks. Phytoplankton were collected in 64-pm nets. They were examined microscopically and concentrated by filtration onto precombusted glass fiber filters. Zooplankton were collected in 150-pm nets. Individuals were examined live under a microscope for removal of detritus and for identification. Cleaned animals were held live in fibered (1pm) seawater before being filtered onto glass fiber filters. Samples were taken throughout the experiment from the three control, 4S, 8S, and 8N tanks. Benthic animals were collected monthly during the experiment from sediment cores and a t the end of the experiment from the entire trays of sediment. Organisms were hand-picked live from sieved sediment, held in filtered (1 pm) seawater overnight for gut clearance, and either filtered whole (small organisms, generally less than 1 mm) or dissected before being dried. All animals were examined microscopically and any visible sediment was removed. Isotopic Analysis. All samples were analyzed by combusting an aliquot to COz in a radio-frequency furnace. The purified gas was measured manometrically (for percent organic carbon) and analyzed on a VG Micromass 602 isotope ratio mass spectrometer. Results are expressed as 613C (per mille) versus the PeeDee Belemnite standard. Analytical variability was f0.3%0,about the same as the precision as established by intercalibrations (24). Details of the methods are given by Gearing et al. (25). Calculations. Calculations of the relative amounts of different carbon sources in a sample are based on a simple mixing equation:
6, = ( f l ) ( 6 1 )+ ( f i ) ( S ~ ) + + ( f J ( 6 n ) (1) where 6, is the isotope ratio of the mixture, f, is the fraction of carbon from source x (x = 1-n), and 6, is the isotope ratio of source x. For sediments where there are only two sources of carbon (fl + f 2 = I), this reduces to the often-used equation (26) 6, = (1- f i ) ( 6 1 ) + ( f i ) ( 6 2 ) (2) For animals, eq 2 must be modified to account for (1)the isotopic fractionation during assimilation by the organism (25) and for (2) changes in the isotope ratio of phytoplanktonic carbon brought about by the presence of sludge or nutrients, Historically the latter have been ignored and the former has been accommodated by introducing a per trophic level shift constant ( T ) of approximately 1%0 unit trophic level difference between organism and food: 6,, = 6, + T ,
where 6,, is the isotope ratio of an organism eating food from a mixture of sources, T, = 6,, - 6,, and 6,, is the isotope ratio of an organism eating only food ultimately from source x. In this paper, the following assumptions have been made. For uptake of new phytoplanktonic production in 296
Environ. Sci. Technol., Vol. 25, No. 2, 1991
the nutrient tanks, we assumed that (1) the isotope ratio of old phytoplanktonic production from before the start of the experiment (6,) was the same as that of phytoplankton in the control tanks, (2) the average isotope ratio of new production throughout the experiment (6,) was representative of the whole experiment, and (3) the newly produced organic carbon was taken up via the normal food web (type FW), i.e., T1 = T,: 6om
+ TI) + f2(62 Ti = Tz = 6,1 - 61
= (1 - f J ( 6 1
+ Tz)
Substituting and rearranging f2
= fneaproduction =
(60m
-
601)/(6Z
(3)
-
For uptake of sludge, it was assumed that the sludge did not change isotopically during up to 3 months in the mesocosms. The isotope ratio of phytoplanktonic production in the sludge tanks (6,) was measured, rather than assumed to be the same as in the control tanks. For uptake via the normal food web (type FW), it was assumed that T1 = T2 = hO1 - fil, giving the equation 60,
fi
= (1- f 2 ) ( 6 1 ( +
= fsludge =
[(6om
Tl) + f 2 @ 2 + T,)
- 601) - (61, - 61)1/('b
-
(4)
For direct uptake (type D) it was assumed that T2 = l % o (25),giving f2
= fsludge = [(6orn - 601) - (61,
-
61)1/[(6,
+ 1)- (61, + 601 - 61)l ( 5 )
Comparison of the similarity of groups was made by using the Mann-Whitney U-Wilcoxon rank sum W test (two-tailed, corrected for ties). Nonparametric statistical tests such as this one do not require the assumption of equal population variances, which are often hard to prove for environmental samples. Separations were accepted as statistically significant when the confidence level was greater than 90%. Results and Discussion Sewage. Weekly samples of sewage sludge were analyzed separately. They contained an average of 3.1 f 1.1% solids ( n = 14) and 40.1 f 3.7% (w/w) organic carbon on a carbonate-free basis (n = 12). Similar values for organic carbon (34.8 f 3.170, n = 24; 34%, n = 2) have been reported for sludge from Los Angeles County (6) and New York City (10). Cranston, RI, sludge had 613C values of -23.5 f 0.4%0 (n = la), virtually identical with those of Los Angeles (6), -23.5 f 0.5%0(n = 24). Two values from New York (-26.0 and -25.7%0)were more negative (10). The isotopic ratios of sludges should be controlled by the input carbon (types of plants and industries) and by the processes carried out during sewage treatment. Water Column. The phytoplankton assemblage in Narragansett Bay and the MERL control mesocosms varies isotopically over the year, with least negative values during diatom blooms in late winter/spring and most negative ratios when productivity is low and nanoplankton predominate in late summer/fall(25). In this experiment, phytoplankton and particulate organic carbon (POC) ratios in the 4 s and 8s sludge mesocosms and in the three control mesocosms followed this pattern (Figure 1A and B), declining slowly from around -20% in June when diatoms were abundant to around -23% in September when the water column was dominated by nanoplankton. Although there was some stimulation of diatom production in the first few weeks of the experiment in the sewage tanks, no
Table I. Average Carbon Isotope Ratios f One Standard Deviation (Number of Samples) in Carbon Sources and Organisms Grouped by Trophic Position, and Calculated Uptake of Sewage Sludge and Fresh Planktonic Production (Percent of Body Carbon) sludge tanks
carbon sources phytoplankton sewage sludge consumers all water column/filter feeders (type 1) 1. Mercenaria mercenaria 2. Ensis directus 3. Mya arenaria 4. Mulinia lateralis 5. Polycirrus eximius 6. Pandora gouldiana 7. zooplankton 8. Crepidula fornicata and plana 9. Ceriantheopsis americanus 10. Petricola phalodiformis 11. Anadara transuersa all suspension feeders (type 2) 12. Chaetozone sp. 13. Polydora ligni 14. Streblospio benedicti 15. Mediomastus ambisetus all deposit feeders (type 3) 16. Pitar morrhuana 17. Nucula annulata 18. Ninoe nigripes 19. Yoldia limatula 20. Maldanid polychaetes 21. Pherusa affinis all predatory feeders (type 4) 22. Eupleura caudata 23. Lumbrinereis fragilis 24. Cerebratulus s p . 25. Nassarius trivittatus 26. Nereis virens 27. Ophioglycera gigantea 28. Nephtys incisa 29. Turbonilla sp.
nutrient tanks
% UpLLIKt. ..-*-I--
control tanks 613C
613C"
-21.6 f 1.2 (15)
-21.5 f 1.6 (15) -23.5 f 0.4 (12)
FWb
(1) f 1.3 (2) f 0.5 (2) f 1.6 (5) (1) f 0.5 (6) f 1.2 (12) f 0.6 (4) f 1.5 (3) f 1.1 (2) f 0.8 (4)
-19.1 -19.0 -19.6 -19.0
f 0.4 (2)
-19.3 -18.6 -18.8 -18.4 -17.1 -17.5
f 1.5 (5) f 1.0 (17)
-18.5 -18.2 -18.7 -17.8 -17.4 -17.7 -16.4 -16.5
(1) f 1.0 (3) (1) (1) f 2.2 (2) f 1.0 (3) f 0.3 (3) f 0.1 (3)
-20.2 -22.5 -22.6 -19.2 -20.0 -21.5 -20.8 -21.4 -21.7
f 2.7 (2)
f 0.1 (2) f 0.5 (4)
(1) f 1.5 (13)
f 0.7 (4) f 0.5 (2) f 1.8 (3) f 1.4* (4)
(1) f 0.5 (2) f 0.5 (4)
f 1.9 f 0.8 f 0.7 f 0.2
(2) (4) (3) (2)
-19.5 -18.7 -18.4 -19.0 -17.7 -18.0
f 1.6 f 0.7 f 0.4 f 0.9
(5) (19) (2) (6) f 0.5 (3) f 0.6 (2)
0 100 100
0
0
0 45 20 60 100 55 55 15 55 21 15 10 0 35 35 30 58
-20.0 f 0.4 (2)
43
0 65 70
45
-20.1 f 0.7 (2) -20.0 f 0.4 (3) -19.8 f 0.4 (5) -20.0 f 0.5* (5)
613CO
FWb
-17.3 f 2.9* (7) 40
-21.5 -21.5 -21.3 -21.3 -20.1 -21.4 -20.1 -20.7 -20.5 -20.3 -19.7
% uptake
Db
80
0
47 19 52 72 26 31 31 10 31 9 9 5 0 17 13 12 32 39
-17.8 f 1.1 (2) -22.7 f 1.0 (2)
0 100
0 100
-17.8 (1) -17.6 f 1.0 (3) -18.2 (1)
10 65 90
4 21 30
-17.1 -16.7 -18.3 -19.6 -17.0 -18.0 -16.2 -18.6 -17.1 -17.6 -18.5
(1) (1) f 1.8 (2) f
1.2* (7)
(1) (1) f 1.3* (6)
f 1.2* (3) f 1.9* (6) f 0.6 (2) f 1.2 (3)
-18.6 -18.0 -16.7 -18.2
f 1.1 (2) f 0.6 (3) f 1.5* (3) f 0.9 (5)
-16.4 -18.0 -19.0 -17.3 -16.9 -17.1
k 0.8* (6) f 1.6 (11)
-16.8 -16.1 -17.6 -16.6 -16.4 -16.0 -15.4
f
(1) f 2.2 (5) f 0.4 (2) f 0.3 (2)
2.1 (2)
f 0.9* (2)
0.1 (2) 2.7 (2) (1) (1) f 0.4* (2) f f
70 100 100 70 40 72 79 91 49 79 63 28 30 12 23 67 19 20 67 14 0 26 5 9 33 40 49 26 28 23 40 23
a An asterisk indicates values significantly different from control values a t the 90% confidence level. *See Experimental Section (calculations) for explanation of and equations used for type FW (food web) and type D (direct).
large, sustained phytoplankton blooms resulted from the addition of sewage sludge (13). Because of the prevalence of nanoplankton during most of this experiment, the isotopic difference between phytoplankton and sludge was quite small. Nevertheless, stable carbon isotope ratios were able to trace the sludge in the sediments and the benthic organisms. In situations when the isotopic difference between phytoplankton and sludge is larger (for Narragansett Bay and the MERL tanks, when larger phytoplankton are blooming), this isotopic tracer should be able to give results with smaller standard errors. A comparison of the isotope ratios for POC from control and sludge tanks showed their time-averaged means to be almost identical (-21.6%0 and -21.5%0, Table I). The sludge tank values could not be distinguished statistically from those of the control tanks a t the 90% confidence level. Only three POC samples from the 8s sludge mesocosm had 613C values close to that of the added sludge. The similarity of results from sludge and control mesocosms is in agreement with conclusions from gravimetric data (13), showing that the sludge did not remain in the water column but sedimented within hours of its addition. Phytoplankton isotope ratios well outside the control mesocosm range were measured in a previous MERL nutrient-enrichment experiment (20). Intense blooms, fer-
tilized by nutrient additions, depleted the amount (and presumably the 12Ccontent) of dissolved inorganic carbon (IOC), this Rayleigh-type isotopic reservoir effect resulting in photosynthetically fixed carbon with ratios of up to -8%. Data from these control and nutrient mesocosms (17, 18) fit the equation
r2 = 0.65
n = 93
where [IOC]oequals 2 X mol of C/L. This empirical relationship is similar to that found for the 613C of IOC on a coral reef (27). Predicted isotope ratios for phytoplankton in the mesocosms during the sludge experiment were determined from this equation and the known [IOC] values, calculated from the temperature, pH, and alkalinity of the water (28). They are shown as the shaded areas in Figure 1. The measured values in control and sludge tanks are similar to the range predicted. In the nutrient tanks, only a few measurements of 8N were made, but these agree well with values predicted from the model. The added nutrient salts caused higher primary production with 13C-enriched isotope ratios, similar to previous results (16, 17). Time-averaged means of phytoplankton for the experiment were Environ. Sci. Technol., Vol. 25, No. 2, 1991
297
-21
-231
160
,
-25,
I
-l3Ic
-154
-174
-19-
-214160
260
240
I 280
JULIAN DATE, 1984 Figure 1. Isotope ratio versus Juiian date of measured particulate organic carbon and phytoplankton (solid circles), measured zooplankton (open circles), and phytoplankton calculated from the concentration of dissolved inorganic carbon (shaded areas) for (A) sludge mesocosms 4s and 8S,(B) three control mesocosms, and (C) nutrient mesocosm 8N. Lines connect phytoplankton values from the same mesocosms.
calculated from measured and predicted values (Table I); the mean for nutrient tanks was significantly less negative than that for controls (-17.3 versus -21.6%0, 98.1% confidence level). Zooplankton, including copepods and polychaete larvae, showed no isotopic evidence of sludge uptake (Figure 1, Table I). Even in the 8s treatment, zooplankton ratios could not be separated statistically from those in control tanks a t the 90% confidence level. Zooplankton assemblages in sludge tanks had higher biomasses and altered species compositions compared to those in control tanks (13), but this was not a result of sludge ingestion or assimilation. Zooplankton in the 8N nutrient tank readily assimilated the fresh planktonic production (separable from controls with 99.9% confidence). Their isotope ratios confirm the high predicted phytoplankton values around days 180-210 (Figure IC). In all tanks, zooplankton 613C values were consistently more positive than those measured and predicted for phytoplankton (by 1.5, 1.5, and 1.1%0 in control, sludge, and nutrient mesocosms, respectively), in line with the expected shift due to trophic position (25). Sediments. Sediments from control, sludge, and nutrient tanks were measured 3 days before the start of the experiment and 65 and 97 days into the experiment. Mesocosm 8s was also sampled 2 weeks after the start. Surface organic carbon contents (2.24 f 0.2170, n = 9) and 6l3C values (-21.6 f 0.4%0,n = 9) were initially similar in all tanks, close to the means reported for previous experiments (25). The only nutrient mesocosm to show changes in 613C values was 8N,which was statistically less negative than control sediments (-21.1 versus -21.5%0) a t the 90% confidence level. The large bloom of diatoms around Julian 298
Environ. Sci. Technol., Vol. 25, No. 2, 1991
200
240
280
JULIAN DATE, 1984 Figure 2. Percent of added sludge carbon found in the sediments over time in 8s (solid circles, total; open circles, 0-1 cm), 4s (triangles, total), and 1s (squares, total) mesocosms.
day 200 (during the first month of the experiment) produced the equivalent of a pulse of isotopically heavy carbon. This was seen in the top 1 cm of sediment from the +65-day sampling as a +1.5%0shift in 613C (to -20.1%0)and a slight (0.3%) increase in total organic carbon. By the end of the experiment, the isotope ratio was only 0.5% more positive than control values and the organic carbon contents had fallen to control levels, showing the rapid utilization of this fresh organic matter. Sewage carbon accumulated in the sediments of the sludge mesocosms (Figure 2). Surface (0-1cm) sediments were significantly different from controls in all three sludge treatments (-22.2 versus -2l.6%, 92.2% confidence level). The clearest isotopic signal was found in the data for mesocosm 8S, which received the most sludge. Isotope ratios of the &l- and 1-2-cm sediments were significantly more negative in the 8s tank than in control microcosms (-22.2 versus -2l.6%, 94.7% confidence level). Half the added sludge was found in the sediments of 8s at the end of the experiment, the ratios having declined from -21.6 to -23.3%0,with percent organic carbon increased from 2.4 to 3.7%, in the top centimeter. An equal percentage of the added sludge was calculated for the sediments of 4S, the values changing from -21.6 to -22.6%. The lesser buildup of sludge in 4 s during the early part of the experiment, if real (values are near the limits for detecting differences), may reflect the ability of the microbial community to respire the smaller quantities of sludge. Measurable accumulation was even slower in the 1s mesocosm, where sludge was not detected isotopically until the end of the experiment (slight shift to -21.7%). Given the large sedimentary reservoir of organic carbon, it is not surprising that organic carbon contents proved less sensitive for detecting sludge inputs than stable isotope ratios. None of the percent organic carbon values measured showed a significant accumulation of sludge carbon except 8s at the end of the experiment, although this latter value (43% of sludge added) agreed well with that calculated from isotopes ratios (49%). Within the sediments, the sludge carbon was mixed down to 2 cm rapidly (within 2 weeks), although the majority remained in the top l cm. Figure 2 compares the amount of sludge in the 0-1-cm level of 8s with the total in the sediment. In all tanks, an average of 62% of the sludge remained in the top 1 cm. During experiments in which petroleum hydrocarbons and radionuclides were added to the mesocosms, the addenda were mixed to 3 cm (23,29); the difference may be one of analytical sensitivity. Once in the sediment, the sewage carbon was not refractory. Calculations of the expected 613C of sediments
. ..*
. ..
defined modes and show no overlap with other modes”. When these control animals are compared with other . * controls and with various treatments, it is convenient to * . 613c-19_1 .* group by feeding type in order to simplify the budget modeling and to have enough samples for statistical -21treatment. However, a more biologically realistic picture J arises from comparison of individual species (Figure 3B-D) even though differences between only a few samples are often not statistically significant. There were small, random isotopic differences between species means of control animals in this experiment and those from all years (Figure 3C). These illustrate the level of natural isotopic variability that normally arises between -5 years, between seasons, and between populations (sampling animals of different sizes and biochemical states). Isotope ratios from the two groups of controls are not statistically different. Both the freshly fixed phytoplanktonic carbon in the nutrient mesocosms (Figure 3D) and the sludge carbon in the sewage mesocosms (Figure 3B) found their way rapidly (
