Estuaries Vol. 27, No. 2, p. 201-208 April 2004

Nutrient Limitation of the Macroalga Enteromorpha intestinalis

Collected along a Resource Gradient in a Highly

Eutrophic Estuary

KRISTA KAMERI*] ~, PEGGY FONG 2, RACHEL Ig~ENNISON 2, a n d KENNETH SGHIFF 1

1 Southern Califer:~da Coastal Water Research Project, 7171 Fenwick Lane, West'minster; Califernia 92683

Department of Organismic Biology, Ecology and Evolution, University of California, Los Angeles, 621 Charles E. Young Drive South, Los Angeles, California 90095-1606

ABSTRACT: We conducted a laboratory exper iment to quantify nutrient (nitrogen and phosphorus ) limitation of ma- croalgae collected along a gradient in water column nutrient availability in Upper Newpor t Bay estuary, a relatively nutrient-rich system in southern California, United States. We collected Enteronmrpha intestiTrali, andwate r fo r use in the exper iment f rom five sites ranging f rom the lower end of the estuary to the head, Initial algal tissue N and P concen- trations and molar N:P ratios---as well as water column NO~ and total Kjeldahl nitrogen (TKN)-- inereased along a spatial gradient f rom the lower end toward the head. Water column soluble reactive phosphorus (SRP) varied among sites as well but did not follow a pat tern of increasing f rom the seaward end toward the head. Algae f rom each site were assigned to one of four experimental treatments: control (C), nitrogen enr ichment (+N) , phosphorus enrichment (+P) , and nitrogen and phosphorus enrichment ( + N + P ) . Each week for 3 wk we replaced the water in each unit with the appro- priate treatnmnt water to mimic a poorly f lushed estuary. After 3 wk, the degree of nutrient limitation of E. intestiTrali, varied spatiallywith distance f rom the head of the estuary. Growth of E. intestiTrali, collected f rom several sites increased with N enrichment alone and increased fur ther when P was added in combination with N. This indicated that N was limiting and that when N was sufficient, P became linfiting. Sites f rom which E. intestiTrali, exhibited nutrient linfitation spanned the range of background water column NO~ (12.9 --- 0.4 to 55.2 --- 2.1 b~M) and SRP (0.8 --- 0.0 to 2.9 --- 0.2 b~M) concentrations. Algae that were N limited had initial tissue N levels ranging f rom 1.18 --- 0.03 to 2.81 --- 0.08% dry weight and molar N:P ratios ranging f rom 16.75 -+- 0.39 to 26.40 -+- 1.98.

In t roduc t ion

With the deve lopmen t of macroalgal b looms in estuaries t h r o u g h o u t the world (McComb et al, 1981; Sfriso et al, 1987; S ch ram m and Nienhuis 1996; Hernf indez et al. 1997), it is critical to un- ders tand the factors regula t ing growth and bio- mass accumulat ion . Macroalgal b looms often oc- cur following increased nu t r i en t loading f rom wa- tersheds (Valiela et al. 1992; Duar te 1995; Nixon 1995; Flindt et al, 1999; S ch ram m 1999) and are p rob lemat ic as excessive p roduc t ion of macroa lgae may reduce the habi ta t quality of an estuary by de- plet ing oxygen levels (Sfriso et al. 1987; Valiela et al, 1992), leading to fish and inver tebra te mortal i ty (Raffaelli et al. 1991) and ul t imately to changes in communi ty s t ructure (Raffaelli et al. 1989; Norkko and g o n s d o r f f 1996; Valiela et al. 1997; g o l a m et al, 2000).

* Con-esponding aufllor; tele: 851/771-4168; e-mail: kkamer@ mlrnl.calstate.edu

]" Cur ren t address: Marine Pollution Studies Laboratory, Moss Landing Marine Laboratories, 7544 Sandholdt Road, Moss Landing, CalKornia 95089.

Macroalgae clearly r e spond to changes in avail- ability of bo th n i t rogen (N) and p h o s p h o r u s (P) (Harl in and Thorne-Mil le r 1981; Valiela et al, 1992; Fong et al. 1998; Peckol et al, 1994). Growth is often st imulated when nu t r i en t supply is in- creased, indicat ing that nut r ients were l imiting (Delgado and Lapoin te 1994; Larned 1998; Lotze and S c h r a m m 2000), as is c o m m o n in mar ine and estuarine systems (Ryther and Dunstan 1971; Han- isak 1983; Howar th 1988; H o l m b o e et al, 1999; Smith et al. 1999),

Study of nu t r ien t l imitat ion of phy top lank ton (Howar th 1988; Rudek et al. 1991; Fisher et al. 1992; H o l m b o e et al. 1999; Tomasky et al, 1999) indicates that the degree and na ture of nu t r i en t l imitat ion may vary spatially within an estuary (Fle- m e r et al, 1998). Water co lumn N and P levels are general ly h igher nea r the head of a system and decrease toward the m o u t h (Rizzo and Christian 1996; H e r n ~ n d e z et al. 1997; Nedwell et al. 2002), which may lead to increased nu t r i en t l imitat ion dora,-estuary. Due to differential rates of nu t r i en t processing (Ryther and Duns tan 1971), the relative a b u n d a n c e of N and P available to p r ima ry pro-

�9 2004 Estuarine Research Federation 201

202 K. Kamer et al.

Fig. 1. Map of Uppe r Newport Bay estuary, California, Unit- ed States, with five sites f rom which E~terornorpha i~testinalisand water were collected for de te rmina t ion of n u t r i e n t l imitation.

ducers may vary along a spatial gradient, generally causing nutr ient limitation to shift f rom P to N as distance from the head of the estuary increases (Doering et al. 1995; Pitkfinen and Tamminen 1995).

In southern California, blooms of Enterom~rpha intestinatis are c o m m o n (Peters et al. 1985; Rud- nicki 1986; Kamer et al. 2001), and there is a need to fur ther unders tand site-specific factors that limit their growth. Several southern California estuaries exhibit gradients in water column nutr ient avail- ability along an axis f rom the head to the mou th (Page et al. 1995; Fong and Zedler 2000) including Upper Newport Bay estuary (UNB; California De- par tment of Fish and Game 1989; Boyle 2002), a highly eutrophic southern California system. Rel- ative availability of water co lumn N and P can dif- fer between head and down-estuary areas (Boyle 2002). The objective of this study was to determine whether N or P limits growth of macroalgae col- lected along an estuarine nutr ient gradient in UNB. We hypothesized that E. intestinatis from down-estuary sites would be nutr ient limited and that the occurrence of nutr ient limitation may de- crease with increasing proximity to the head of UNB. We tested for possible limitation by both N

and P to d e t e r m i n e if the l imi t ing n u t r i e n t changed with increasing distance from the head.

Materials and M e t h o d s

We conducted a laboratory exper iment to test whether N or P limited growth of E. intestinaSs from five sites in UNB. Sites ranged from the lower end of the estuary (Site 1) to the head (Site 5), where San Diego Creek, the pr imary surface water nutr ient source (U.S. Environmental Protect ion Agency [EPA] 1998), enters UNB (Fig. 1). At each site, we collected algae fi-om exposed intertidal mudflat and water fi-om mid-water depth ( - 1 m) on June 11, 2001, on a flood tide. Materials were transported back to the laboratory within 5 h. In the laboratory, E. intestinatis from each site was cleaned of debris and other organisms and placed in shallow pans filled with aerated water from the cor responding site. Pans were kept outdoors over- night in a temperature controlled water bath (20 _+ 2~ The collected water was placed in the dark in a 6~ cold room until added to experimental units.

O n June 12, 2001, a por t ion of the water f rom each site was divided into 4 aliquots to create the experimental treatments (control [C], n i t rogen enr ichment [+N] , phosphorus enr ichment [ +P] , and n i t r o g e n and p h o s p h o r u s e n r i c h m e n t [ +N +P]) , resulting in a three-factor experimental design: site X N enr ichment X P enr ichment . Con- trol treatments were ambient water from each site with no additions. Nutr ient a m e n d m e n t treat- ments were enriched with NO s and P O 4 t o increase N and P concentrat ions by 400 and 40 bM, re- spectively, over background levels. Enr ichment concentrat ions were based on known levels of NO s in UNB (Boyle 2002; Kamer unpubl ished data). P was added in a 10:1 N:P ratio based on the work of Boyle (2002) showing a year.long water column N:P grand mean of 10.4:1. Due to collection of water during a flood tide, water column nutr ient concentrat ions (Table 1) were considerably lower than values measured at low tide in other studies of UNB (Boyle 2002; Kamer unpubl ished data) or other southern California estuaries (Page et al.

TABLE 1. M e a n ( + S E ) b a c k g r o u n d w a t e r c o l u m n n u t r i e n t c o n c e n t r a t i o n s a t the t i m e o f co l l ec t i on f r o m e a c h o f the 5 sites in UNB. n = 3. S u p e r s c r i p t s a - e d e n o t e m e a n s t h a t a re s ign i f i can t ly d i f f e r e n t f r o m e a c h o t h e r (p < 0.05, F i she r ' s LSD fo l lowing s i g n i f i c a n t 1-I=actor ANOVA) . Values b e l o w d e t e c t i o n l im i t s (8.57 p,M for N a n d 1.61 p,M fo r P) were set to h a l f the d e t e c t i o n lkni t .

W~.ter Column 1,1utrienta (bM) Salinity

Site (psu) NO~ NH 4 SRP

1 ,87 12.9 (0 .4) " 6.2 (1.2) b 0.8 (0 .0) " 92.9 (8 .4) " 2 35 28.3 (1.2) b 25.7 (1.2) a 2.9 (0.2) b 95.2 (38.3)" 3 ,33 36.2 (0.6) ~ 12.6 (1.4) ~ 1.1 (0.5) ~ 100 (14.9) ~ 4 31 55.2 (2.1) e 17.4 (2.7) c 2.5 (0.3) b 238.1 (41.7) b 5 33 42.4 (0.6) d 1.79 (0.0) ~ 0.8 (O.Op 302.4 (56.9) b

1995; Fong and Zedler 2000). Over the course of a year, Boyle (2002) measu red h igher m e a n water co lumn nut r ien t concent ra t ions near the head of the estuary, p roximal to this study's Site 5 (158- 800 ham NO~; 4.8-16.7 ham total P) relative to down-estuary areas near Site 1 (5-90 p.M NOs; 1.8- 11.5 poM total P), and Kamer (unpubl i shed data) also found a g rad ien t in water co lumn NOs in UNB of 414 bdV[ at Site 5, 101 ~M at Site g, and 49 bdV[ at Site 1 2 mo before the cur ren t study.

E. i,ztesti,zMis f rom each site was placed in nylon mesh bags and spun in a salad spinner for 1 mi- nu te to r emove excess water. Algae were wet weighed and 8.0 -- 0.1 g sub-samples were added to glass exper imenta l units (1.5 1 total vo lume) , each containing 800 ml of the appropr i a t e solu- tion. Five addi t ional sub-samples of algae were pro- cessed for initial tissue N and P analyses. Experi- men ta l units were placed in a r andomized array ou tdoors in a t empe ra tu r e control led water ba th (20 -+ 2~ and covered with window screening to reduce inc ident light (2,200-9,500 >moles m z s at midday) by - 3 0 % to s imulate coastal levels (1,405- 1,956 IJ, moles m -2 s -z, Arnold and Murray 1980). Replicat ion was five-fold with the except ion of the site g Cont ro l and site g +N t reatments , which only had three replicates each due to not having col- lected enough algae f rom that site. T h e r e were a total of 96 units. Salinity was m o n i t o r e d every 9 d with a hand-he ld r e f r ac tome te r and deionized wa- ter was added to compensa te for evaporat ion. Sa- linity was main ta ined within 2 psu of initial levels measu red for each site at the t ime of collection.

T h e e x p e r i m e n t ran for 8 wk with weekly ex- change of t r ea tmen t water to simulate low turnover characteristic of poor ly f lushed estuaries in south- ern California (Zedler 1982, 1996; Largier et al. 1997; Fong and Zedler 2000). Weekly exchange is appropr i a t e based on hydrodynamic models of UNB showing that 55% of a dissolved, conservative tracer r ema ined in the bay for 3 d following a s torm flow of 100 cfs (Resource M a n a g e m e n t As- sociates, Inc. 2008), which is considerably h igher than no rma l base flow condi t ions (Public Facilities and Resources D e p a r t m e n t 2001). Units were re- r andomized in the water ba th each week.

At the end of the exper iment , algae were re- moved f rom each unit and wet weighed after be ing spun in nylon mesh bags in a salad sp inner for 1 rain. G r o w t h was d e t e r m i n e d as the p e r c e n t change f rom initial wet biomass. Each sample was rinsed briefly in freshwater to remove external salts, dried in a forced air oven at 60~ to a con- stant weight, and g round with m or t a r and pestle for subsequent tissue N and P analysis. N and P conten t of algae are r epor ted as concen t ra t ion (% dry weight).

Nutrient Limitation of Estuarine Macroalgae 203

Water samples were filtered (Whatman GF/C) pr ior to be ing frozen for subsequen t analysis. Wa- ter co lumn N O s was r educed to NO~ via cadmium reduct ion; NO~ was measured spec t ropho tomet r i - cally after diazotat ion (Switala 1999; Wendt 1999). Water co lumn NH4 was hea ted with solutions of salicylate and hypochlor i te and de t e rmined spec- t rophotomet r ica l ly (Switala 1999; Wendt 1999). Water co lumn total Kjeldahl n i t rogen (TKN, which is all fo rms of N except N O s and NO~) was deter- mined by the wet oxidat ion of n i t rogen using sul- furic acid and digestion catalyst. T h e p r o c e d u r e converts organic n i t rogen to NH4, which is subse- quent ly de t e rmined (Carlson 1978). Water co lumn soluble reactive p h o s p h o r u s (SRP) was de t e rmined spec t rophotomet r ica l ly following react ion with am- m o n i u m molybdate and an t imony potass ium un- der acidic condit ions (American Public Hea l th As- sociation 1998). These a u t o m a t e d me thods have detect ion limits of 3.57 poM for all fo rms of N and 1.61 poM for R Values of samples below detect ion limits were set to half the detect ion limit to allow statistical analysis of data.

Algal tissue N was d e t e r m i n e d using an induc- tion furnace and a the rmal conductivity detector (Dumas 1981). Algal tissue P was d e t e r m i n e d by a tomic absorp t ion spec t romet ry (AAS) and induc- tively coupled p lasma a tomic emission spec t rome- try (ICP-AES) following a nitric ac id-hydrogen per- oxide microwave digestion (Meyer and Keliher 1992). Algal tissue N:P ratios were calculated on a mola r basis.

All data were tested for normal i ty and h o m o g e - neity of variance; no t ransformat ions were needed. Differences in initial water nut r ien t concent ra t ions (NOs, NH4, TKN, SRP) were analyzed by 1-factor AVOVA (site). Multiple pairwise compar i son pro- cedures (Fisher's Least Significant Difference test [LSD]) were employed following significant AN- OVA (p < 0.05) to de t e rmine differences a m o n g individual sites. A m o n g t r ea tmen t differences in growth, tissue nu t r i en t concentra t ions , and tissue N:P ratios were analyzed using three- fac torANOVA (site X N e n r i c h m e n t X P enr ichment ) . Significant interact ions did no t occur unless otherwise noted. Following a significant ANOVA, mult ip le compar- isons were used to de t e rmine differences a m o n g individual t rea tments within factors (Fisher 's LSD).

Resu l t s

Nutr ien t concent ra t ions of water collected J u n e 11, 2001, varied with site (ANOVA, p < 0.001 for N O > NH4, and SRP, p - 0.005 for TKN). NOs was lower in water f rom the m o r e seaward sites (Sites 1 and 2) c o m p a r e d to the sites fu r ther up-estuary, and the highest NOs levels were found in water f rom site 4 (Table 1). N H 4 and SRP were variable

204 K. Kamer et al.

TABLE 2. Mean (+SE) initial Erderorz~orptaa intestinalisfissue ni- trogen and phosphorus concentrations and molar N:P ratios t-ore each of the 5 sites in UNB. n 5. superscripts a-d denote means that are significantly different from each other (p < 0.05, Fisher's LSD following significant 1-tZactor ANOVA).

Tissue iXlutriez?t CoI?teI?t (as % dry weight)

Site N P Molar N:P

1 1.18 (0.03) 4 0.156 (0.002) ~ 16.75 (0.39) ~ 2 1.47 (0.06) b 0.164 (0.007) ~ 20.09 (1.87) b 3 2.21 (0.03) ~ 0.230 (0.003) b 21.26 (0.26) b 4 2.81 (0.13) d 0.238 (0.009) b 26.40 (1.98) ~ 5 2.62 (0.08) a 0.320 (0.009) c 18.16 (0.40) ~,b

a m o n g sites a n d d id n o t i n c r e a s e wi th p r o x i m i t y to San D i e g o Creek . TKN was s imi la r in w a t e r f r o m Sites 1-3 a n d h i g h e r f rom Sites 4 a n d 5 (Tab le 1). Tissue N a n d P c o n t e n t o f E. intestinatis i n c r e a s e d a l o n g a spa t ia l g r a d i e n t f rom the l ower e n d of the e s tua ry t oward the h e a d (Table 2). Tissue N:P mo- lar r a t ios r a n g e d f rom 16.75 to 96.40 (Tab le 2) a n d i n c r e a s e d fi-om Site 1 to Site 4.

A f t e r 3 wk, g r o w t h o f E. intestinalis was signif i- can t ly a f f ec t ed by si te (ANOVA p - 0.0001). W i t h the e x c e p t i o n o f Site 5, g r o w t h i n c r e a s e d with p r o x i m i t y of c o l l e c t i o n sites to t he h e a d of the Bay (Fig. 9; Si tes 1-4) . E. intestinMis g r o w t h was a f f ec t ed by N e n r i c h m e n t (ANOVA p - 0.0001) b u t n o t by P e n r i c h m e n t (ANOVA p - 0 .6968). C o m p a r e d to con t ro l s , g r o w t h of a lgae f i o m Sites 1, 2, a n d 4 i n c r e a s e d in t he N e n r i c h m e n t a l o n e t r e a t m e n t s (F i she r ' s LSD p < 0.05 for C ve rsus + N at each s i te) , i n d i c a t i n g tha t N was l imi t ing . T h e ef fec t o f N e n r i c h m e n t on g rowth was s t r o n g e s t for a lgae f r o m the d o w n e s tua ry sites. G r o w t h of a l g a e f r o m Site 1 e n r i c h e d wi th N was 14.8 -- 2.3% g r e a t e r t h a n g r o w t h o f con t ro l s ; a lgae fi-om Site 2 e n r i c h e d with N g rew 16.3 -- 2.0% m o r e t h a n con t ro l s . T h e s e va lues were n o t d i f f e r e n t (F i she r ' s LSD p - 0 .657), b u t a lgae fi-om Site 2 e n r i c h e d with N g rew m o r e re la t ive to c o n t r o l s t h a n a lgae fi-om site 4 e n r i c h e d with N did (8.3 _+ 9 .4%; F i s h e r ' s LSD p - 0.097).

T h e i n c r e a s e in b i o m a s s o f E. intestinalis f r o m Site 3 w h e n N was a d d e d c o m p a r e d to c o n t r o l s was s ta t is t ical ly i n s ign i f i c an t (F i she r ' s LSD p - 0.054) as va r i ab i l i ty in the c o n t r o l t r e a t m e n t was re la t ive ly h i g h a n d r e p l i c a t i o n o f the c o n t r o l a n d + N t rea t - m e n t s was th ree - fo ld . T h e r e was no s ign i f i can t dif- f e r e n c e in g r o w t h of a lgae f r o m site 5 b e t w e e n con- t rol a n d + N t r e a t m e n t s ( F i s h e r ' s LSD p - 1.000), which , a l o n g wi th t he r e s p o n s e o f a lgae f rom Site 3, r e s u l t e d in a n i n t e r a c t i o n b e t w e e n site a n d N e n r i c h m e n t (ANOVA p - 0.0024).

F o r E. intestinatis co l l ec t ed f r o m Sites 1, 2 a n d 4, g r e a t e r g r o w t h w h e n P was a d d e d in c o m b i n a t i o n with N (Fig. 2; F i s h e r ' s LSD p < 0.005 for + N vet-

Fig. 2. Enteromorpha intestinalis biomass (as % change from initial) grown with ambient (Control), nitrogen enr ichment (+N), phosphorus enr ichment (+P), or nitrogen and phos- phorus enr ichment (+N+P) solutions. Bars represent +1 SE.

sus + N + P at e ach site) r e s u l t e d in an i n t e r a c t i o n b e t w e e n N a n d P e n r i c h m e n t (ANOVA p - 0.0039). Th i s i n d i c a t e d tha t P b e c a m e l i m i t i n g w h e n N l i m i t a t i o n was re l i eved . G r o w t h o f E. intes tinalis fi-om Site 5 was less w h e n P was a d d e d , re- su l t ing in an i n t e r a c t i o n b e t w e e n site a n d P en- r i c h m e n t (ANOVA p - 0 .0001).

T h e r e was a g r a d i e n t in E. intestinatis f inal t issue N c o n c e n t r a t i o n across sites (ANOVA p - 0.0001) in all t r e a t m e n t s (Fig. S). To s o m e d e g r e e these d i f f e r e n c e s a r e a r e f l e c t i o n of the g r a d i e n t in ini- t ial t issue N c o n c e n t r a t i o n s (Tab le 2) wi th the ex- c e p t i o n o f a lgal t issue N f r o m Site 4. W h e n N was a d d e d , f inal t issue N levels of a lgae f rom Sites 1 a n d 2 were 39.7 _+ 2.8% a n d 21.0 _+ 2.3% ( m e a n + SE for + N a n d + N + P t r e a t m e n t s , n - 10) h igh - er t h a n in i t ia l levels, respect ively . T i s sue N o f a lgae f rom site 4 was 22.6 -+ 1.1% lower, a n d for a lgae fi-om Sites S a n d 5, f inal t issue N was w i th in 6% of in i t ia l levels.

T i s sue N was s ign i f ican t ly a f f e c t e d by N e n r i c h - m e n t (ANOVA p - 0.0001) b u t n o t by P e n r i c h - m e n t (ANOVA p - 0.2878). Fo r all sites, t issue N c o n c e n t r a t i o n was g r e a t e s t w h e n N was a d d e d re- ga rd l e s s of P a d d i t i o n . T h e r e was an i n t e r a c t i o n b e t w e e n site a n d N e n r i c h m e n t ( A N O V A p - 0.002) p r o b a b l y d u e to a s l ight ly h i g h e r c o n c e n t r a - t ion of t issue N in t he + N versus + N + P t r e a t m e n t s f rom Site 4.

E. intestinMis f inal t issue P c o n c e n t r a t i o n was sig- n i f i can t ly a f f e c t e d by site a n d P e n r i c h m e n t (AN- OVA p - 0.0001 for b o t h fac tors) b u t n o t by N e n r i c h m e n t (ANOVA p - 0 .4489). F o r all sites, tis- sue P c o n c e n t r a t i o n was g r e a t e s t w h e n P was a d d e d r e g a r d l e s s o f N a d d i t i o n (Fig. 3). T i s sue P c o n c e n - t r a t ions in every e x p e r i m e n t a l t r e a t m e n t were

3.0

2.5

g '~ 2.0

Z

1~0 . -

0.35

0 3 0 _c ._~

0,25

"1@ 0,20

r, .~ 0 1 5

~- 010

0.05 ff

C, 00

Fig. 8.

Conlrol r - - ' l + N NN{N + P I + N + F '

1 2 3 4 5

S i t e

Enteromorpha intestinalis tissue nitrogen and phospho- rus concentration as % dry wt after 3 wk in ambient (Control), nitrogen enrichment (+N), phosphorus enrichment (+P), or nitrogen and phosphorus enrichment (+N+P) solutions. Bars represent + 1 SE.

greatest f rom Site 5. This was likely due to high initial tissue P concentra t ion (Table 2) combined with only modera te growth relative to the algae from the other sites, and caused an interaction be- tween site and P enr i chment (ANOVA p - 0.0038). In the treatments no t enr iched with P, tissue P of algae increased among algae collected along the nutr ient gradient in UNB, possibly reflecting the initial algal tissue P gradient (Table 2) similar to tissue N. In treatments where P was added, tissue P was similar among algae collected from different sites within UNB with the exception of algae from Site 5.

E. intestinMis tissue N:P ratios were significantly affected by N enrichment , P enr ichment , and site (ANOVA p < 0.0001 for each factor) and therewas significant interaction between N and P enrich- ment (A_NOVA p < 0.0001). Lowest N:P ratios oc- curred in treatments receiving P enr i chment alone (Table g) followed by + N + P treatments. The high- est ratios were in the +N only treatments. There was significant three-way interaction (ANOVA p < 0.0001) due to different patterns in tissue N:P ra-

Nutrient Limitation of Estuarine Macroalgae 205

TABLE 3. Mean (• final molar N:P ratios o f Er~tero~r~orpt~a intestinalis tissue f rom all experimental treatments.

Treatrneot

give Control + N +P + N + P

1 22.70 (o.45) 52.01 (1.80) 7.77 (o.23) 13.98 (0.19) 2 26.44 (0.29) 44.58 (1.27) 8.58 (0.21) 15.11 (0.27) 3 29.89 (0.16) 43.94 (1.16) 11.76 (0.24) 16.85 (0.21) 4 52.55 (0.58) 45.62 (0.99) 12.99 (0.29) 18.71 (0.50) 5 25.27 (0.40) 38.94 (0.83) 10.93 (0.16) 17.09 (0.34)

dos among sites within each experimental treat- ment.

D i s c u s s i o n

Nutr ient limitation of E. intestinalis from UNB varied spatially th roughou t the bay. The degree of ni t rogen limitation increased with distance from the head of the estuary. Algae from down-estuary sites, where background water column NOs con- centrations were lowest, were more N-limited than E. intestinalis fi-om Site 4, which had the highest background water co lumn NO s concentrat ion. Wa- ter from Site 4 had one of the highest background water co lumn TKN levels, yet algae were still N lim- ited in spite of this potential source of N (Probyn and Chapman 1989; Hanisak 198S; Navarro-An- gulo and Robledo 1999; Lotze and Schramm 2000; Tyler et al. 9001). There was no indication of N limitation of algae fi-om Site 5, but growth of algae from this site was relatively moderate . Site 5 was closest to the mou th of San Diego Creek, which is known to contain selenium, heavy metals, organic pesticides, and PCBs (EPA 2009). These com- pounds may have inhibited algal growth.

Once N limitation was relieved, P limitation oc- curred in algae from several sites, regardless of wa- ter column or tissue P availability. Similarly, Del- gado and Lapointe (1994) also observed P limita- tion of two species of tropical, fleshy macroalgae once N limitation was relieved. Lapointe (1987) found increases in Gracilaria tikvakiae growth with either N or P enr ichment and synergistic interac- tion between the two nutrients, and Schaffelke and Klumpp (1998) used tissue nutr ient concentra- tions and estimated nutr ient requirements to infer that both N and P limited Sargassum baccutaria growth, In several instances, it has been found that phytoplankton abundance was most limited by N and that P became limiting when N supply in- creased (McComb et al, 1981; Taylor et al. 1995),

Initial tissue N concent ra t ion was not always an effective predictor of nutr ient limitation over the experimental period. N limitation occurred in E. intestinatis with initial tissue N concentra t ions that varied by more than two-fold at the beginning of the experiment. N limitation may have been ex-

2 0 6 K. Kamer et al.

pected in algae from Sites I and 2, which had ini- tial tissue N concentra t ions < 2% dry weight, the suggested critical concentra t ion below which ma- croalgal maximal growth rates are limited by inter- nal N concentra t ion (Birch et al. 1981; Hanisak 1983; O 'Br ien and Wheeler 1987; Pedersen and Borum 1996). N limitation of algae from Site 4 occurred as well. Algae fi-om this site had initial tissue N concentra t ions of - 2 . 8 % dry weight, above which fur ther increases in N supply should not stimulate increased growth. Algae became lim- ited as growth reduced internal stores over the 3- wk experiment. In some treatments, tissue N con- centrations decreased over the course of the ex- periment, indicating that algae were using internal stores of N as well as water column supplies to sus- tain growth.

Nei ther initial no r final tissue molar N:P ratios of E. intestinalis were consistently good indicators of nutr ient limitation. Earlier work suggests that macroalgal tissue molar N:P ratios < 16 indicate N limitation, ratios between 16 and 24 indicate that N and P are present in sufficient supply relative to each other, and ratios > 24 indicate P limitation (Bjgrnsgter and Wheeler 1990; Wheeler and Bjgrn- sgter 1992). In our experiment, algae from Site 4 with the highest initial N:P ratio of ~26 displayed N limitation when P limitation may have been pre- dicted based on tissue nutr ient content. After 11 to 12 d, Larned (1998) found N limitation of Utva fasciata with an initial tissue N:P ratio of 48.3:1. Our end of exper iment tissue N:P ratios of E. in testinalis from control treatments were also gener- ally in the range (>24) of P limitation (Bj6rnsgter and Wheeler 1990), yet the algae from Sites 1, 2, and 4 were N limited. These data in combinat ion suggest that algal tissue N:P ratios indicative of nu- trient limitation may vary over regional or local scales. They may be site- and species-specific, in- dicating a need for direct experimental determi- nat ion of nutr ient limitation rather than relying on tissue N:P ratios alone.

Several of our results were more in agreement with earlier work (Bj6rnsgter and Wheeler 1990). N:P ratios of E intestinalis in +N only treatments (>38) and +P only treatments (<13) were consis- tent with the Bj6rnsgter and Wheeler paradigm as algae from Sites 1, 2, and 4 became P-limited when N was supplied and remained N-limited when P was supplied.

The results of our exper iment demonstra te spa- tial variability in one species of macroalgae in one season. Future investigation of nutr ient limitation of macroalgae in southern California should in- clude other dominan t macroalgal species such as U/va, and seasonal manipulat ions should be per- formed to determine the temporal extent of nu-

trient limitation. Incorpora t ion of sediments into experimental units should be considered in order to better approximate field condit ions and obtain more realistic insight into nutr ient limitation of macroalgae, or nutr ient limitation experiments could be done in situ. Additional efforts should also be made to de termine how prevalent patterns of nutr ient limitation are across estuaries in the southern California region. Increased unders tand- ing of factors that control seasonal algal b looms is vital to efforts to prevent them.

ACKNOWLEDGMENTS

Th i s work was partially f u n d e d by the Santa Aria Regional Water Quali ty Control Board (State Water Resources Control Board Contrac t #00-181-180-0) a n d by the Uni ted States Envi- r o n m e n t a l Protect ion Agency (project #021925 m R Fo n t ) . All nu t r i en t analyses were conduc t ed by the D e p a r t m e n t o f Agri- culture and Natural Resources Analytical Laboratory, University of Califonaia, Davis. We t h a n k the Califonaia D e p a r t m e n t o f Fish and Game and J o h n Scholl for access m UNB, a n d Lauri Green, Andre ia win iams , Dario Diehl, Risa Cohen , Caitlin Fong, an d J im Kamer for he lp in the lab a n d field. Th ree a n o n y m o u s re- viewers provided valuable c o m m e n t s and sugges t ions for im- proving the quality of this manuscr ip t .

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Subrnitted, January& 2003 Revised, May 14, 2003 Accepted, June 3, 2003