The influence of ammonia on the growth and photosynthesis of

Hydrobiologia 275/276: 219-231, 1994. E. Mortensen et al. (eds), Nutrient Dynamics and Biological Structure in Shallow Freshwater and Brackish Lakes. © 1994 Kluwer Academic Publishers. Printed in Belgium.

219

The influence of ammonia on the growth and photosynthesis of Ruppia drepanensis Tineo from Dofiana National Park (SW Spain) L. Santamaria, C. Dias & M. J. M. Hootsmans InternationalInstitute for Infrastructural,Hydraulic and Environmental Engineering(IHE), P.O. Box 3015, 2601 DA Delft, The Netherlands Key words: Ruppia drepanensis, ammonia toxicity, temperature effects, photosynthesis, growth Abstract In a laboratory experiment, Ruppia drepanensisTineo seedlings from a brackish marsh in Southern Spain were grown at 20 and 30 C, at three different nitrogen levels. These levels were obtained by the addition of a slow release fertilizer (23 % NH 4 NO 3 by weight) to a sediment mixture of sand and clay (3:1). Several morphometric parameters were recorded during the first five weeks of the experiment, and photosynthesis and respiration were measured after 7 weeks of growth. Results showed a significant reduction of growth and development with increasing nitrogen and temperature levels. Dark respiration increased strongly at high nitrogen levels. At the same time, net photosynthesis at 250 and 500 ,E m- 2 s- , Pm, Km and LCP were not affected by either factor. We attribute these phenomena to ammonia toxicity, since relatively high total ammonia (NH 3 + NH+ ) levels were found in the interstitial water. Introduction The effects of increasing nutrient loads on aquatic ecosystems have been described relatively well. It seems clear that there is a causal relation between increasing periphyton and algal blooms, and macrophyte decline and disappearance (e.g. Phillips et al., 1978; De Nie, 1987). But apart from changes in the dominant primary producers through shading effects, high nitrogen levels in the water and sediment may also directly affect the physiology and thus the growth dynamics of submersed macrophytes through toxic effects of ammonia (Mattes & Kreeb, 1974). The aim of this research was to study the effect of different temperatures and high nitrogen levels on the growth, development and photosynthesis of the submerged macrophyte Ruppia drepanensis Tineo under laboratory conditions. This species is the dominant aquatic plant in the brackish area

of the Dofiana marsh (Southern Spain). This wetland is surrounded by agricultural land used for rice culture and other cereal crops. The high use of fertilizers there can be expected to cause an increase in the concentration of nitrogen inside the marsh area. We hypothesized that flowering may be triggered by decreased nitrogen availability, and in that case increased nitrogen levels could postpone flowering and reproduction and lead to a longer growth period. Although there was no direct support in literature for this assumption, it seemed a reasonable mechanism. If this should in fact occur in a temporary wetland like the Dofiana brackish waterbodies, where aquatic macrophytes survive the dry period by producing droughtresistant seeds, population survival of these annual macrophytes could be threatened. Related to the temperature effect, we expected plant development to be faster at higher tempera-

220 tures (Barko & Smart, 1981; Barko et al., 1982), together with earlier flowering and reproduction (Richardson, 1980). We also expected an increase in the rates of net photosynthesis and respiration with increasing temperature, as reviewed in Hootsmans & Vermaat (1991).

Materials and methods Experimental setup: Plant material cultivation Seeds attached to plant material were collected in an old channel of the Salinas de San Isidoro (Dofiana National Park, SW Spain), at the end of the growing season (June 1991). A mixed population of R. drepanensis and Althenia orientalis (Tzvelev) Garcia-Murillo & Talavera occurs in this locality. Seeds and wet plant material were stored together in plastic bags at 4 C, until October 1991. The seeds were germinated in tap water, in the dark and at room temperature (20 oC). After three days, germinated seedlings were selected and transferred to containers with brackish water (0.5 g 1- 1 artificial sea salt), where they stayed for one week under low light conditions (10 to 20 pE m- 2 s- ) and at room temperature. Previous experience had shown that this treatment resulted in the best survival percentage. After one week the seedlings, of between 3 and 5 cm size, were randomly distributed over the treatments and subsequently planted. Two levels of temperature (20 + 0.5 and 30 _ 0.5 C) and three levels of nitrogen (referred from now on as 'NO', 'N1' and 'N2') were combined in six treatments. Each treatment consisted of one aquarium (60 x 40 x 40 cm 3 ), with the exception of the 'NO' nitrogen level that had two aquaria for each temperature. The eight aquaria were placed in two phytotrons. The aquaria had independent pumping systems for cooling and heating, and a common light system. Temperature was maintained at the required levels by a system of two pumps with independent thermostats, one connected to a cooling system and one equipped with a heating system. To en-

sure that the water was homogeneously mixed, the heating pump was working continuously, although not constantly heating. Demineralised water was used to replenish the aquaria when necessary. As a light source 17 fluorescent tube lamps (Philips, colour 84, 36 W) were used in each phytotron, providing a light intensity of 240 BE m- 2 s- at 1 cm below the water surface (measured with a LICOR LI-192S sensor). The photoperiod was 16 hours. Each aquarium contained 39 plastic cups (8 cm height) filled with 100 ml of a mixture of sand and clay (3:1 by weight) and covered by 1 cm of washed sand. Space among the cups was also filled with washed sand. The remaining volume of the aquaria was filled with tap water one week before planting the seedlings. The three levels of nitrogen were obtained by adding a slow release fertilizer (Osmocote® pellets) to the sediment mixture. The pellets contained 23 % of ammonium nitrate by weight, and

were placed at 1 and 3 cm above the bottom of the cups. The clay used in the sediment mixture and clay samples from Dofiana National Park (lucio Vetas Altas Chico, July 1991) were analyzed to determine the total nitrogen content (Novozamsky etal., 1983). The values obtained (clay used in the sediment mixture: 938 + 434 mg N kg- ', average+ standard deviation; Dofiana clay: 1592 mg N kg-') were used to determine the three nitrogen treatments. Nitrogen level N1 was comparable to the nitrogen content of Dofiana clay (108.8 mg N per sediment cup), NO was half of it (54.4 mg N/cup), and N2 had twice as much (228 mg N/cup). Samples of surface and interstitial water were taken for analysis of alkalinity and total ammonia (NH 3 + NH, ) 1, 2 and 4 weeks after starting the experiment. Two replicate samples were taken in polyethylene flasks. The interstitial water was sampled each time from different, randomlyselected plant cups, using a semipermeable stick (70 x 3 mm) connected to a 10 ml syringe (RHIZON SSS, Rhizosphere Research Products, NL). Interstitial water samples were taken

221 overnight. Alkalinity was determined by titrimetry with 0.02 N sulphuric acid, using methyl orange and phenolphthalein as end-point indicators (APHA, 1985). Total ammonia was determined by potentiometric measurements (METHROM Titroprocessor 636 dosimeter, ORION 95-10 specific electrode).

Characterizationof plant material During the first five weeks of the experiment the following parameters were recorded in 20 randomly-selected plants from each treatment: number of shoots, number of leaves per shoot, length of the largest leaf and number of flowers per plant. The same plants were measured each week. In the 'NO' treatment, 10 different plants were randomly selected from each of the two aquaria at each temperature treatment. After four weeks, six plants were harvested from each treatment to determine their biomass (above and below-ground fractions). Fresh weight and dry weight (70 C, 24 h) were recorded. Plants collected after 7 weeks for the photosynthesis measurements were also used for biomass determination (fresh, dry and ash-free dry weights, the last after 3 h at 520 C). Subsamples were taken to determine the aboveground to belowground biomass ratio. Plants from the treatment '30 C, N2' could not be used for biomass measurements, due to their very small size. Ash free dry weight of the 4-weeks plant material was calculated from the dry weight values by using the dw/afdw regression line fitted using the 7-weeks plant material. Experimental set-up for photosynthesis measurements All photosynthesis measurements were done in a 96 1 aquarium connected to a cooling system which kept the temperature during the incubation at + 1 ° C of the desired value. Together with this system, three heating coils where used for the measurements at 30 C. The temperature selected

was the same as that at which the plants did grow (20 and 30 C respectively). The aquarium was filled with tap water and 20 g NaHCO3 was added to arrive at saturating inorganic carbon levels (3.7 mM HCO£; Sand-Jensen, 1983). Nitrogen was bubbled in the aquarium prior to the oxygen production measurements, in order to reduce the concentration of dissolved oxygen. Three independent, replicate systems were used. Each one consisted of an electrode chamber and a 5 cm diameter perspex tube interconnected with pvc tubing. A peristaltic pump (Watson Marlow 504U) was used to circulate the water. Temperature and dissolved oxygen were recorded every 10 seconds with a Campbell 21X datalogging set connected to three WTW EO-196 oxygen electrodes and WTW OXY 196 electrode meters. Light was provided by a Philips 400 W HPIT metal halide lamp. Different light levels were created by varying the distance between lamp and water surface and using a neutral density net. A shallow perspex flow-through waterbath was suspended beneath the lamp to absorb the infrared radiation. Flow rate was 0.75 1 min-' (equivalent to 6.5 mm s- 1 in the perspex tube). This is about half of the flow rate used by Hootsmans & Vermaat (1991) and Sand-Jensen (1983). Nevertheless, since in a comparable incubation chamber Westlake (1967) found that rates of photosynthesis of Potamogeton pectinatus L. did not increase further above flow rates of 0.4 mm s - , we expect that the used flow rate had no appreciable limiting effect on photosynthesis. Five to six randomly-selected, intact plants of seven weeks old were used per tube. Plants were carefully washed out of the sediment, and kept overnight in darkness in order to permit acclimation and to deplete lacunar oxygen reserves. Next morning, dark respiration of the plant material was measured during 30 to 45 minutes. Subsequently, the tubes were exposed to the different light levels (15 to 45 min, until at least a 0.5 mg 1-' increase in dissolved oxygen concentration was reached), starting with the lowest intensity and ending with the highest. Between each measurement the tubes were opened and the medium

222 inside was completely replenished with the surrounding water. As the light field could not be held homogeneous for the light levels above 150 ME m- 2 s - 1, we measured light on three points along each tube and used the average intensity per tube for further calculations. Light intensities at 1 cm below the water surface outside the tubes were on average 25, 50, 75, 100, 150, 190,265, 300, 380 and 435 #E m- 2 s- I respectively. For N1 and N2 plant material, only respiration and production at 265 and 435 ME m-2 s- were measured. It was not possible to measure a PI curve for the plants grown at 30 C and nitrogen level N2. Due to their very low biomass, these plants did not produce a sufficiently large increase in oxygen concentration during the time available for the measurements.

Calculations and statistical analysis Morphometrical data Statistical analysis of the morphometrical data followed Vermaat & Hootsmans (1991), with minor modifications, and were done with the SAS statistical package (SAS Institute Inc., 1988). An exponential curve was fitted for the morphometric variables using a non-linear iterative technique based on the Marquardt algorithm (Conway et al., 1970), since the inflexion point of a logistic growth curve was not reached during the experimental time. To facilitate computation, four curves per aquarium were fitted, each one with data from five, randomly-selected plants. Parameters of the curves were compared by means of an Analysis of Variance (ANOVA) followed by multiple comparisons among treatments. ANOVA tests were done with the General Linear Models (GLM) procedure, after loglo transformation if residuals were not normally distributed. Homogeneity of residual variances was checked with a plot of predicted versus residual values. For multiple comparisons, we used the LSMEANS option in SAS. Comparisonwise error

rates (CER) for each comparison were adjusted to maintain an experimentwise error rate (EER) of 0.05.

Photosynthesis data Each data set relating oxygen concentration to time for a particular light level was checked for measurement errors, and lag phases were excluded. Oxygen exchange rates were calculated by linear regression and expressed in g per g afdw of plant tissue per minute (g 02 g afdw- ' min - ').

The resulting data set for each treatment replica consisted of the experimental light levels and the corresponding 02 exchange rates. A rectangular hyperbola (Michaelis-Menten model) was fitted to each data set using the non-linear regression (NLIN) procedure of the SAS statistical package (SAS Institute Inc., 1988). The resulting model parameter estimates were used for ANOVA and multiple comparisons (as above), together with one derived parameter, the light compensation point (LCP). Estimated net oxygen production rates at 250 and 500 pE m - 2 s-1 (Pe250 and Pe500) were also used, as sometimes the estimated maximum rate of gross photosynthesis (parameter Pm) was reached beyond the light level range used in the experiment. A total of 6 light-response (PI) curves was obtained from the experiment, for the plants grown at the zero nitrogen level (3 replicate curves at 20 C, and 3 at 30 C). For comparison with other PI curves from literature (see Discussion), only these curves will be used. PI curves measured for the plants grown at the nitrogen levels N1 and N2 were based on the respiration rate and 2 production rates (at 265 and 435 ME m-2 s- light intensities). Two different data sets were used for the statistical analyses: the first one included the measured respiration and photosynthetic rates (RESP, P265 and P435), and the second one included the parameter estimates (Pm, Km and R) and the production rates (Pe250 and P500) estimated with the fitted curves.

223 Results Water chemistry Initial bicarbonate alkalinity of the tap water used to fill the aquaria was 1.2 mM. Bicarbonate alkalinity decreased over time in all aquaria, from initial values of about 0.9 mM (st week) to values of 0.4 to 0.7 mM. Carbonate alkalinity was not detectable in any of the samples. Total ammonia concentration in the surface water showed a general increase with increasing nitrogen level (Table 1), but it never exceeded 0.5 mg NH2 -N 1-' (except in one case, the first week in the '30 C, N2' aquarium). Total ammonia concentration in the interstitial water showed a more clear increase with increasing nitrogen level. NH -N ranged from 0.5 to 3.5 mg 1- for the NO treatments (at both temperatures), 5 to 40 mg 1-1 for the N1 treatments, and above 15 mg 1- 1 (but extremely variable at 20 C, from 9 to 300 mg 1- ) for the N2 treatments.

A Two Way ANOVA test was done to compare the effects of growth temperature (GTEMP) and nitrogen level (NITR) on the estimated relative growth rates of the variables 'number of leaves' (RLV), 'number of shoots' (RSH) and 'length of the largest leaf (RLL). GTEMP and NITR effects were significant for all three parameters tested. The interaction of nitrogen and temperature was also significant for RLV and RSH. All three relative growth rates decreased with increasing temperature and increasing nitrogen level (Fig. 1). Multiple comparisons were done among the 9 combinations of GTEMP and NITR (comparisons for interaction between both factors are excluded; EER=0.05, CER=0.0056). For RLV, all comparisons except 'NO vs N1, 20 C' and 'N1 vs N2, 20 C' were significant. For RSH and RLL, a significant nitrogen level effect was only present between NO and N2 at both temperatures. Temperature effect was sig-

I

Plant morphometry Only a few plants flowered in some of the treatments during the 7 weeks experiment. Thus, the parameter 'number of flowers per plant' was not included in the statistical tests. Table 1. Ammonia concentration in the water column and porewater (mg NH4 -N 1- ). Results shown in the table are the average value of two replicate samples (*: no replicate samples). NO, N1 and N2 represent the three nitrogen levels.

SH

//

LG

('3

"0

a)

4--' c

0.10

('M

I'3

¢0

0.05

a)

Porewater

Weeks:

1st

2nd

4th

1st

2nd

4th

20 20 20 20 30 30 30 30

0.11 0.08 0.09 0.23 0.10 0.06 0.14 1.96

0.12 0.09 0.12 0.42 0.35 0.07 0.13 0.18

0.06 0.07 0.38 0.27 0.28 0.16 0.37

3.5* 1.1 14.4 297.9 2.3 1.6 4.9 24.9

0.9 2.9 9.8 9.6 1.4 1.1* 4.4 17.1

0.6 1.9 39.5 235.0 3.0 2.8 11.0 22.0

C, NO C, NO C, N1 °C, N2 C, NO C, NO C, N1 C, N2

E

0.15

Characterizationof plant material

Water column

LV

>

cc a)

fT'

0.00 T2NO T2N1 T2N2 T3NO T3N1 T3N2

Fig. 1. Means and standard deviations of relative growth rates (day - 1) of the number of leaves (LE, filled bars), number of shoots (SH, densely hatched bars) and the length of the longest leaf (LG, thinly hatched bars). T2, T3 represent the temperatures 20 and 30 C, NO, N1 and N2 represent the three nitrogen levels.

224 nificant in all comparisons but one (N1 level of RSH) for all three parameters tested. The derived parameters number of leaves, number of shoots and largest leaf length on the 30th day of age (LV30, SH30 and LL30, respectively) were calculated with the fitted curves. Two Way ANOVA (after loglo transformation) yielded significant results for both growth temperature and nitrogen level (p < 0.001), as well as their interaction (p