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Inhibition of nitrogen fixation in salt marshes measured by acetylene reduction
Estuarine
and
COaStal
l%fUYi?Ze
SCienCe
(1974)
2,
301-305
Notes and Discussions Inhibition of Nitrogen Fixation in Salt Marshes Measured by Acetylene Reduction0 Charlene D. Van Raalte,” Ivan Carpenter’ and John M. Teal’
Valiela,”
Edward
J.
bBoston University Marine Program, Marine Biological Laboratory, Woods Hole, Mass., 02543 and ‘Woods Hole Oceanographic Institution, Woods Hole Mass. 02543, U.S.A. Received 6 February I974
Nitrogen fixation as estimated by acetylene reduction takes place in intertidal sand and mud in salt marshes and is inhibited by additions of nitrogenous compounds such as sewage sludge or urea. Fixers on the marsh use combined nitrogen available from other sources in preference to nitrogen fixation. Marshes can remove substantial amounts of nitrogen from eutrophic waters by this shift in the nitrogen cycle.
Introduction The nitrogen cycle in the marine environment is poorly understood yet is particularly important since nitrogen is a main limiting nutrient (Ryther & Dunstan, 1971; Goldman et al., 1973). Estuarine areas such as salt marshes are being increasingly contaminated with nitrogenous
compounds
from
sewage
outfalls,
agricultural
fertilizer
and
cesspool
seepage
(Ketchum, 1972). Such enrichments may have effects on the nitrogen cycle. Nitrogen fixation is probably important in salt marshes since nitrogen fixing blue-green algae and bacteria are abundant. The extent of nitrogen fixation in marshes and the effect of enrichment by nitrogenous compounds upon fixation rates is unknown. In laboratory cultures, nitrogen fixation and the abundance of heterocysts in blue-green algae can be reduced by adding nitrogen containing compounds, but the effect of combined nitrogen on fixation measured in the field remains unclear (Fogg, 1949; Dugdale & Dugdale, 1965 ; Stewart, 1969; Horne & Fogg, 1970). We have been conducting a study of the effects of sewage sludge and urea enrichment on Great Sippewissett salt marsh, Massachusetts, since 1971. Within the marsh, there are two habitats where fixers are found. The largest is the surface of the marsh under the grass canopy. Seaward is the blue-green algal mat, a flat expanse over sand and devoid of higher vegetation.
Methods We added fertilizer (IO'& N, 6% P,Os, 4% K,O), made (Kerr-McGee Corp.), every other week to salt marsh plots of drainage ditches in Great Sippewissett Marsh, Cape November during 1972 and 1973, in a high dosage of 25-2 “Contribution
No.
3138
from
the
301
Woods
Hole
commercially from sewage sludge m in radius located at the head Cod, Massachusetts from May to g/mz/week (HF) and a low dosage IO
Oceanographic
Institution.
302
C. Van Raalte et al.
of 8.4 g/ms/week (LF). There were two replicate plots for each dosage. Details on the chemical composition of the sludge fertilizer are given elsewhere (Valiela et al., 1973). Two other plots were treated with urea fertilizer (46% N by weight) at a dosage of 5.6 g/m2/week. Nitrogen fixation was measured by acetylene reduction (Stewart, 1971) on triplicate cores (0.5 cm2 x 0.5 cm deep) from each plot. Since the plots were separated by as much as 200 m, there was substantial variability among the parts of the marsh where the plots were located. To account for this variability, triplicate cores were taken 5 to IO m outside of each plot to act as controls. The fertilization effects did not extend beyond the edges of the plots (Valiela & Teal, 1974). After incubation in Is-ml serum bottles for two hours in situ, ethylene production was measured by flame ionization gas chromatography on a Hewlett Packard gas chromatograph with a 0.3 cm x 2.7 m Poropak R column. The moles of ethylene were converted to moles of ammonia using a factor of 1-5 (Stewart et al., 1968). To relate fixation rates to levels of ammonia nitrogen in sediment water, samples of pore water from the top IO cm of marsh sediment were obtained at ebbing tide by removing a core 6 cm in diameter and collecting water percolating into the core hole. The concentration of NH, N was measured by the method of Salorzano (1969). To evaluate the effect of sludge and urea enrichments on nitrogen fixation on the bluegreen algal mat, we established eight I ms plots, two each treated with the sludge fertilizer at the LF and HF dosage, two with the urea dosage and two controls. The plots were located at least 5 m apart to reduce mutual contamination. The sampling and gas chromatographic analysis were identical to those described for the marsh surface.
Results Nitrogen fixation was measured during the summer months of 1972 and 1973 (Tables I and The heterocystous blue-green algae, Cdothrix contarenii and purple photosynthetic bacteria are known fixers and are found in the marsh and mat. The blue-green Lyngbya aesturii also comprises the algal mat and may be a fixer. Fertilization with the high dosage of sewage sludge significantly inhibited nitrogen fixation in the salt marsh surface in both years (Table I). The low level of sludge fertilizer had no clear effects on fixation rates. Fertilization with urea significantly decreased the fixation of nitrogen in 1973. This suggests that the addition of nitrogenous compounds is responsible for the inhibition of nitrogen fixation, rather than being the result of toxic effects from other substances in the sewage fertilizer (Horne & Goldman, 1974). The standard error of the means are substantial, demonstrating the heterogenous distribution of algae (Estrada et al., 1974). The inhibitory effect of combined nitrogen enrichment can also be seen in Figure I which shows the relationship between nitrogen fixation rates and the amount of NH, N in the pore water. We use the O-IO cm sample since preliminary measurements show that there seems to be no consistent gradient in NH, N in the pore water between the surface and a depth of IO cm. High fixation rates may or may not occur at low NH, N concentrations. This may reflect algal response to patchy NH, concentrations at a scale much smaller than that measured in our water samples. Nevertheless, fixation is inhibited as the NH, N concentrations in the pore water increases. The lower fixation rates observed in August 1972 relative to June 1972 (Table I) were probably caused by the increased shading due to grass growth. We have measurements of fixation at midnight compared to mid-day which show that about 98.5% is light dependent 2).
Nitrogen
fixation
inhibition
in salt
marshes
303
(unpublished results). Shading by the grass canopy must therefore be of importance determining fixation rates. I. Algal nitrogen fixation in ng of N/cm2/h marsh plots treated with the high (HF) and low fertilizer (U). TABLE
Outside
Inside June
HF LF
106.8k39.3 224’2 192.6
0
9.2 zt4’3 22.9119’3 100.7i26.3
MaY HF LF U
161.ok34.5
74.7 f5.6 46.0 zt34.5 X09.0 &60.8 230.0&o 92.0&o
Inside
3.8 & 1.6 21.5*1.8 19’6+5’0 3.6f1.2
5.5 NS
1973
28.7k5.6 155.0*86.2 119~of69~0
inside and outside salt dosage of sludge and urea
(Z&S.E.)
(LF)
August 100** 92** 79 NS
“/o Reduction”
1972
100** 100** 30 NS 36 NS
0 0
13.6ho.5 2.3 i 1.3
June 1973
*
40.2f17.2
63.2f5.6 23.0&o
Outside
1972
186k30.3 121f19.1
“6 Reduction
E* -7oNS -89NS 73** 75"'
*
-83.9 142’3 zt37.4 78.8 + IO.9
29’9 f23’9 23.418.9 264.6 i 24’9
s”.y* -7oNS
130.6&16.4 463.2k14.9 14.8rk1.9
168.513'7 3'9iO.S
- 23NS 99**
1.850
88**
293'5
in
There are two plots under each treatment and three replicate samples taken inside and outside of each plot. The outside samples in each plot are the controls for that specific plot. Double asterisks indicate significance at the 0.01 level of a t test between inside and outside means. Single asterisks indicate significance at the 0.1 level. Non-significance is shown as NS. O-I “O,& reduction computed as = IOO where 0 is the rate for the outside ( 0 1 (control) samples and I is the rate for the experimental samples.
Nitrogen fixation was similarly inhibited in all the fertilized blue-green algal mat plots, including the urea treatments (Table 2). This suggests that here also the inhibition of nitrogen fixation is due to the additions of combined nitrogen in the algal mat as was the case in the marsh surface. The algal mat shows greater susceptibility to the additions of fertilizer, responding even to the LF dosage.
0 NH4N
Figure I. Nitrogen fixation fertilized and control plots.
(pgat/O plotted
in sediment
against
water
NH,,
N concentration
in pore
water
of
304
C. Van Raalte et al.
Discussion The amount of nitrogen fixed seemsto be significant to the vegetation and probably contributesto the high productivity of salt marshes.Taking a rough averagevalue of IOO ng/cm2/h for May-July, we calculate that enough nitrogen is fixed (1.2 g N/m”) to account for the nitrogen contained in maximum standing crops in the unfertilized plots (1-5 g N/m2) (Valiela & Teal, 1974). 2. Algal nitrogen fixation in ng of N/cm*/h (.?&s.E.) in two replicate plots in the blue-green algal mat treated with a high (HF) and low (LF) dosage of sludge and urea (U) fertilizers as compared to control plots.
TABLE
Treatment May
I973
HF
ng of N/cme/h
0h Reduction” 100**
0
100**
LF U Control July
1973
HF LF U Control
6.39 lo.62 Ij~Io~6~08 19.80f6.64 5’07 k2.27 7I.go-cs.67 86.90 f2.36 2.17io.04 2’57 ho’70 3’09iO.40 3’52*0’1 I 2.57 fo.08 2.40 ho.20 4.4’30 ho,80 33.56*I.o9o
Double asterisks indicate significance means at the 0.01 level. Single asterisk
91** 81* 75* 93** 94* 93* 92* 9I* 93* 94* -
of a 1 test between indicates significance
treatment at the 0.1
and control level.
‘Seefootnoteto Table I. Even though nitrogen fixation is lowered in the fertilized plots, measurementsof chlorophyll, algal productivity and bacterial oxygen uptake show that there is no decreasein algal photosynthesisor bacterial respiration (Estrada et al., 1974; Van Raalteet al., in preparation; Teal et al., in preparation). This must mean that the micro-organismsare finding other nitrogen sources,perhapsobtaining nitrogen directly from the added fertilizer. Through the use of fertilizer nitrogen, the bacteria and algaewould avoid the energy-requiring fixation processesand still achieve growth rates asgreat or greater than those obtained with fixation asthe main nitrogen source. The ability of salt marshmicro-organismsto usevaried sourcesof combined nitrogen may be involved in making salt marsheseffective removers of nitrogen from coastal waters (Valiela et al., 1973). Additions of combined nitrogen, such as may occur through sewage contamination, may therefore change the structure of the nitrogen cycle in a salt marsh environment, replacing nitrogen fixation as a nitrogen input. This work was supported by NSF Grants GA-28365, GA-29272, GU-3846 and GA-37993. N. Corwin performedthe NH, N Analysis. S. Golubic helped to identify the blue-greenalgae, K. Bums provided help with the gas chromatographic analysis, and K. Smith reviewed the manuscript.
Nitrogen
fixation
inhibition
in salt
marshes
305
References Dugdale, V. & Dugdale, R. D. 1965 Nitrogen metabolism of Lakes III Tracer studies of the assimilation of inorganic nitrogen sources. Limnology and Oceanography IO, 53-57. Estrada, M., Valiela, I. & Teal, J. M. 1974 Concentration and distribution of chlorophyll in fertilized plots in a Massachusetts salt marsh. rournal of Experimental Biology and Ecology 14, 47-56. Fogg, G. E. 1949 Growth and heterocyst formation in Anabaena cylindrica. Annals of Botany 13, 241~ 259. Goldman, F. C., Tenore, K. R. & Stanley, H. I. 1973 Inorganic nitrogen removal from waste water; effect on phytoplankton growth in coastal marine waters. Science, New York x80,955-956. Horne, A. J. & Fogg, G. E. 1970 Nitrogen fixation in some English lakes. Proceedings of the Royal Society of London B172, 351-366. Horne, A. J. & Goldman, C. F. 1974 Suppression of nitrogen fixation by blue-green algae in a eutrophic lake with trace additions of copper. Science, New York 183,409-41 I. Ketchum, B. H. (ed.) 1972 The Waters Edge. MIT Press, Cambridge, Massachusetts. Ryther, J. H. & Dunstan, W. M. 1971 Nitrogen, phosphorous and eutrophication in the coastal marine environment. Science, New York 171, 1008-1012. Salorzano, L. 1969 Determination of ammonia in natural waters by the phenolhypochlorite method. Limnology and Oceanography 14799-801, Stewart, W. D. P. 1969 Biological and ecological aspects of nitrogen fixation by free-living microorganisms. Proceedings of the Royal Society of London B172,367-388. Stewart, W. D. P. 1971 Nitrogen fixation in the sea. In Fertility in the Sea (Costlow, J. E., ed.) Gordon and Breach, New York. pp. 537-564. Stewart, W. D. P., Fitzgerald, G. P. & Burris, R. H. 1968 Acetylene reduction by blue-green algae. Archives fur Mikrobiologie 62, 336-348. Teal, J. M., Smith, K. L. & Valiela, I. Respiration of salt marsh surface communities and the effect of experimental additions of sewage sludge. In preparation. Valiela, I., Teal, J. M. & Sass, W. 1973 Nutrient retention in salt marsh plots experimentally fertilized with sewage sludge. Estuarine and Coastal Marine Science I, 261-269. Valiela, I. & Teal, J. M. 1974 Nutrient limitation in salt marsh vegetation. In Ecology of Halophytes (Reimold, R. J. & Queen, W. H., eds) Academic Press, New York, Van Raalte, C., Valiela, I. & Teal, J. M. Production in benthic salt marsh algae. In preparation.
and
COaStal
l%fUYi?Ze
SCienCe
(1974)
2,
301-305
Notes and Discussions Inhibition of Nitrogen Fixation in Salt Marshes Measured by Acetylene Reduction0 Charlene D. Van Raalte,” Ivan Carpenter’ and John M. Teal’
Valiela,”
Edward
J.
bBoston University Marine Program, Marine Biological Laboratory, Woods Hole, Mass., 02543 and ‘Woods Hole Oceanographic Institution, Woods Hole Mass. 02543, U.S.A. Received 6 February I974
Nitrogen fixation as estimated by acetylene reduction takes place in intertidal sand and mud in salt marshes and is inhibited by additions of nitrogenous compounds such as sewage sludge or urea. Fixers on the marsh use combined nitrogen available from other sources in preference to nitrogen fixation. Marshes can remove substantial amounts of nitrogen from eutrophic waters by this shift in the nitrogen cycle.
Introduction The nitrogen cycle in the marine environment is poorly understood yet is particularly important since nitrogen is a main limiting nutrient (Ryther & Dunstan, 1971; Goldman et al., 1973). Estuarine areas such as salt marshes are being increasingly contaminated with nitrogenous
compounds
from
sewage
outfalls,
agricultural
fertilizer
and
cesspool
seepage
(Ketchum, 1972). Such enrichments may have effects on the nitrogen cycle. Nitrogen fixation is probably important in salt marshes since nitrogen fixing blue-green algae and bacteria are abundant. The extent of nitrogen fixation in marshes and the effect of enrichment by nitrogenous compounds upon fixation rates is unknown. In laboratory cultures, nitrogen fixation and the abundance of heterocysts in blue-green algae can be reduced by adding nitrogen containing compounds, but the effect of combined nitrogen on fixation measured in the field remains unclear (Fogg, 1949; Dugdale & Dugdale, 1965 ; Stewart, 1969; Horne & Fogg, 1970). We have been conducting a study of the effects of sewage sludge and urea enrichment on Great Sippewissett salt marsh, Massachusetts, since 1971. Within the marsh, there are two habitats where fixers are found. The largest is the surface of the marsh under the grass canopy. Seaward is the blue-green algal mat, a flat expanse over sand and devoid of higher vegetation.
Methods We added fertilizer (IO'& N, 6% P,Os, 4% K,O), made (Kerr-McGee Corp.), every other week to salt marsh plots of drainage ditches in Great Sippewissett Marsh, Cape November during 1972 and 1973, in a high dosage of 25-2 “Contribution
No.
3138
from
the
301
Woods
Hole
commercially from sewage sludge m in radius located at the head Cod, Massachusetts from May to g/mz/week (HF) and a low dosage IO
Oceanographic
Institution.
302
C. Van Raalte et al.
of 8.4 g/ms/week (LF). There were two replicate plots for each dosage. Details on the chemical composition of the sludge fertilizer are given elsewhere (Valiela et al., 1973). Two other plots were treated with urea fertilizer (46% N by weight) at a dosage of 5.6 g/m2/week. Nitrogen fixation was measured by acetylene reduction (Stewart, 1971) on triplicate cores (0.5 cm2 x 0.5 cm deep) from each plot. Since the plots were separated by as much as 200 m, there was substantial variability among the parts of the marsh where the plots were located. To account for this variability, triplicate cores were taken 5 to IO m outside of each plot to act as controls. The fertilization effects did not extend beyond the edges of the plots (Valiela & Teal, 1974). After incubation in Is-ml serum bottles for two hours in situ, ethylene production was measured by flame ionization gas chromatography on a Hewlett Packard gas chromatograph with a 0.3 cm x 2.7 m Poropak R column. The moles of ethylene were converted to moles of ammonia using a factor of 1-5 (Stewart et al., 1968). To relate fixation rates to levels of ammonia nitrogen in sediment water, samples of pore water from the top IO cm of marsh sediment were obtained at ebbing tide by removing a core 6 cm in diameter and collecting water percolating into the core hole. The concentration of NH, N was measured by the method of Salorzano (1969). To evaluate the effect of sludge and urea enrichments on nitrogen fixation on the bluegreen algal mat, we established eight I ms plots, two each treated with the sludge fertilizer at the LF and HF dosage, two with the urea dosage and two controls. The plots were located at least 5 m apart to reduce mutual contamination. The sampling and gas chromatographic analysis were identical to those described for the marsh surface.
Results Nitrogen fixation was measured during the summer months of 1972 and 1973 (Tables I and The heterocystous blue-green algae, Cdothrix contarenii and purple photosynthetic bacteria are known fixers and are found in the marsh and mat. The blue-green Lyngbya aesturii also comprises the algal mat and may be a fixer. Fertilization with the high dosage of sewage sludge significantly inhibited nitrogen fixation in the salt marsh surface in both years (Table I). The low level of sludge fertilizer had no clear effects on fixation rates. Fertilization with urea significantly decreased the fixation of nitrogen in 1973. This suggests that the addition of nitrogenous compounds is responsible for the inhibition of nitrogen fixation, rather than being the result of toxic effects from other substances in the sewage fertilizer (Horne & Goldman, 1974). The standard error of the means are substantial, demonstrating the heterogenous distribution of algae (Estrada et al., 1974). The inhibitory effect of combined nitrogen enrichment can also be seen in Figure I which shows the relationship between nitrogen fixation rates and the amount of NH, N in the pore water. We use the O-IO cm sample since preliminary measurements show that there seems to be no consistent gradient in NH, N in the pore water between the surface and a depth of IO cm. High fixation rates may or may not occur at low NH, N concentrations. This may reflect algal response to patchy NH, concentrations at a scale much smaller than that measured in our water samples. Nevertheless, fixation is inhibited as the NH, N concentrations in the pore water increases. The lower fixation rates observed in August 1972 relative to June 1972 (Table I) were probably caused by the increased shading due to grass growth. We have measurements of fixation at midnight compared to mid-day which show that about 98.5% is light dependent 2).
Nitrogen
fixation
inhibition
in salt
marshes
303
(unpublished results). Shading by the grass canopy must therefore be of importance determining fixation rates. I. Algal nitrogen fixation in ng of N/cm2/h marsh plots treated with the high (HF) and low fertilizer (U). TABLE
Outside
Inside June
HF LF
106.8k39.3 224’2 192.6
0
9.2 zt4’3 22.9119’3 100.7i26.3
MaY HF LF U
161.ok34.5
74.7 f5.6 46.0 zt34.5 X09.0 &60.8 230.0&o 92.0&o
Inside
3.8 & 1.6 21.5*1.8 19’6+5’0 3.6f1.2
5.5 NS
1973
28.7k5.6 155.0*86.2 119~of69~0
inside and outside salt dosage of sludge and urea
(Z&S.E.)
(LF)
August 100** 92** 79 NS
“/o Reduction”
1972
100** 100** 30 NS 36 NS
0 0
13.6ho.5 2.3 i 1.3
June 1973
*
40.2f17.2
63.2f5.6 23.0&o
Outside
1972
186k30.3 121f19.1
“6 Reduction
E* -7oNS -89NS 73** 75"'
*
-83.9 142’3 zt37.4 78.8 + IO.9
29’9 f23’9 23.418.9 264.6 i 24’9
s”.y* -7oNS
130.6&16.4 463.2k14.9 14.8rk1.9
168.513'7 3'9iO.S
- 23NS 99**
1.850
88**
293'5
in
There are two plots under each treatment and three replicate samples taken inside and outside of each plot. The outside samples in each plot are the controls for that specific plot. Double asterisks indicate significance at the 0.01 level of a t test between inside and outside means. Single asterisks indicate significance at the 0.1 level. Non-significance is shown as NS. O-I “O,& reduction computed as = IOO where 0 is the rate for the outside ( 0 1 (control) samples and I is the rate for the experimental samples.
Nitrogen fixation was similarly inhibited in all the fertilized blue-green algal mat plots, including the urea treatments (Table 2). This suggests that here also the inhibition of nitrogen fixation is due to the additions of combined nitrogen in the algal mat as was the case in the marsh surface. The algal mat shows greater susceptibility to the additions of fertilizer, responding even to the LF dosage.
0 NH4N
Figure I. Nitrogen fixation fertilized and control plots.
(pgat/O plotted
in sediment
against
water
NH,,
N concentration
in pore
water
of
304
C. Van Raalte et al.
Discussion The amount of nitrogen fixed seemsto be significant to the vegetation and probably contributesto the high productivity of salt marshes.Taking a rough averagevalue of IOO ng/cm2/h for May-July, we calculate that enough nitrogen is fixed (1.2 g N/m”) to account for the nitrogen contained in maximum standing crops in the unfertilized plots (1-5 g N/m2) (Valiela & Teal, 1974). 2. Algal nitrogen fixation in ng of N/cm*/h (.?&s.E.) in two replicate plots in the blue-green algal mat treated with a high (HF) and low (LF) dosage of sludge and urea (U) fertilizers as compared to control plots.
TABLE
Treatment May
I973
HF
ng of N/cme/h
0h Reduction” 100**
0
100**
LF U Control July
1973
HF LF U Control
6.39 lo.62 Ij~Io~6~08 19.80f6.64 5’07 k2.27 7I.go-cs.67 86.90 f2.36 2.17io.04 2’57 ho’70 3’09iO.40 3’52*0’1 I 2.57 fo.08 2.40 ho.20 4.4’30 ho,80 33.56*I.o9o
Double asterisks indicate significance means at the 0.01 level. Single asterisk
91** 81* 75* 93** 94* 93* 92* 9I* 93* 94* -
of a 1 test between indicates significance
treatment at the 0.1
and control level.
‘Seefootnoteto Table I. Even though nitrogen fixation is lowered in the fertilized plots, measurementsof chlorophyll, algal productivity and bacterial oxygen uptake show that there is no decreasein algal photosynthesisor bacterial respiration (Estrada et al., 1974; Van Raalteet al., in preparation; Teal et al., in preparation). This must mean that the micro-organismsare finding other nitrogen sources,perhapsobtaining nitrogen directly from the added fertilizer. Through the use of fertilizer nitrogen, the bacteria and algaewould avoid the energy-requiring fixation processesand still achieve growth rates asgreat or greater than those obtained with fixation asthe main nitrogen source. The ability of salt marshmicro-organismsto usevaried sourcesof combined nitrogen may be involved in making salt marsheseffective removers of nitrogen from coastal waters (Valiela et al., 1973). Additions of combined nitrogen, such as may occur through sewage contamination, may therefore change the structure of the nitrogen cycle in a salt marsh environment, replacing nitrogen fixation as a nitrogen input. This work was supported by NSF Grants GA-28365, GA-29272, GU-3846 and GA-37993. N. Corwin performedthe NH, N Analysis. S. Golubic helped to identify the blue-greenalgae, K. Bums provided help with the gas chromatographic analysis, and K. Smith reviewed the manuscript.
Nitrogen
fixation
inhibition
in salt
marshes
305
References Dugdale, V. & Dugdale, R. D. 1965 Nitrogen metabolism of Lakes III Tracer studies of the assimilation of inorganic nitrogen sources. Limnology and Oceanography IO, 53-57. Estrada, M., Valiela, I. & Teal, J. M. 1974 Concentration and distribution of chlorophyll in fertilized plots in a Massachusetts salt marsh. rournal of Experimental Biology and Ecology 14, 47-56. Fogg, G. E. 1949 Growth and heterocyst formation in Anabaena cylindrica. Annals of Botany 13, 241~ 259. Goldman, F. C., Tenore, K. R. & Stanley, H. I. 1973 Inorganic nitrogen removal from waste water; effect on phytoplankton growth in coastal marine waters. Science, New York x80,955-956. Horne, A. J. & Fogg, G. E. 1970 Nitrogen fixation in some English lakes. Proceedings of the Royal Society of London B172, 351-366. Horne, A. J. & Goldman, C. F. 1974 Suppression of nitrogen fixation by blue-green algae in a eutrophic lake with trace additions of copper. Science, New York 183,409-41 I. Ketchum, B. H. (ed.) 1972 The Waters Edge. MIT Press, Cambridge, Massachusetts. Ryther, J. H. & Dunstan, W. M. 1971 Nitrogen, phosphorous and eutrophication in the coastal marine environment. Science, New York 171, 1008-1012. Salorzano, L. 1969 Determination of ammonia in natural waters by the phenolhypochlorite method. Limnology and Oceanography 14799-801, Stewart, W. D. P. 1969 Biological and ecological aspects of nitrogen fixation by free-living microorganisms. Proceedings of the Royal Society of London B172,367-388. Stewart, W. D. P. 1971 Nitrogen fixation in the sea. In Fertility in the Sea (Costlow, J. E., ed.) Gordon and Breach, New York. pp. 537-564. Stewart, W. D. P., Fitzgerald, G. P. & Burris, R. H. 1968 Acetylene reduction by blue-green algae. Archives fur Mikrobiologie 62, 336-348. Teal, J. M., Smith, K. L. & Valiela, I. Respiration of salt marsh surface communities and the effect of experimental additions of sewage sludge. In preparation. Valiela, I., Teal, J. M. & Sass, W. 1973 Nutrient retention in salt marsh plots experimentally fertilized with sewage sludge. Estuarine and Coastal Marine Science I, 261-269. Valiela, I. & Teal, J. M. 1974 Nutrient limitation in salt marsh vegetation. In Ecology of Halophytes (Reimold, R. J. & Queen, W. H., eds) Academic Press, New York, Van Raalte, C., Valiela, I. & Teal, J. M. Production in benthic salt marsh algae. In preparation.