Springer 2006
Hydrobiologia (2006) 553:121–127 DOI 10.1007/s10750-005-9896-y
Primary Research Paper
Nitrogen and phosphorous excretion rates by tubificids from the Prahova River (Romania) Carmen Postolache*, Geta Rıˆ ßs noveanu & Angheluta˘ Va˘dineanu Department of Systems Ecology and Sustainable Development, University of Bucharest, Spl. Independentei 91–95, 76201, Bucharest, Romania (*Author for correspondence: Fax: 401-411-23-10; E-mail:
[email protected]) Received 11 August 2004; in revised form 19 April 2005; accepted 1 June 2005
Key words: excretion rates, nitrogen, phosphorous, tubificids
Abstract Nitrogen and phosphorous exchange at the water–sediment interface is controlled both by complex physico-chemical factors and biological processes. Zoobenthos excretion is one of the most important processes in the mineralization of sedimented organic mater. In polluted freshwaters, tubificid worms are among the dominant components of the benthic community. Rates of ammonium and inorganic phosphate excretion by tubificids were experimentally assessed. They were related to the tubificid abundance in a stream ecosystem polluted with municipal and industrial wastewater. The relationship between these rates and temperature were investigated within the range of 4–23 C. Relatively constant excretion rates were obtained for both nutrients in the first 8 h of excretion, ranging between 0.076 and 0.226 lg N mg d.w.)1 h)1 and 0.0065–0.01 lg P mg d.w.)1 h)1, respectively. Q10 values of 2.52 for ammonium and 1.31 for phosphate were calculated. If we presume that all excreta eventually enters the water column, then we can calculate that these invertebrates potentially add 39.17 mg N m)2 day)1 and 0.49 mg P m)2 day)1. These values accounts for 17.16 and 7.56% of the nutrient load in the river water, respectively.
Introduction The Danube is the second largest river in Europe. It has a catchment area of 807,000 km2 that stretches over 13 states. Eutrophication of the Danube Basin is a key environmental concern that has emerged in the last few decades, as scientists and managers have observed sharp increases in nitrogen and phosphorous concentrations in freshwater lakes and rivers throughout the region. A management plan for the preservation of good water quality is critically needed, but any such plan must be based on detailed knowledge of nutrient emission sources, pathways and sinks in the catchment area.
Nitrogen and phosphorous exchange at the water–sediment interface is controlled by many complex physico-chemical factors, as well as by biological processes (Bostrom et al., 1982). Zoobenthos can influence nutrient dynamics at the water–sediment interface through excretion of nutrient compounds, and through continuous release of nutrients from sediments through channels created by bioturbation activity (Fukuhara & Yasuda, 1985, 1989; Fukuhara & Sakamoto, 1987, 1988; Rıˆ ßs noveanu et al., 2002, 2004)). There is relatively little known about the effects of macroinvertebrates on nutrient mineralization and exchange processes during bioturbation, particularly in stream ecosystems.
122 The assessment of the role of macroinvertebrates in nutrient cycling in aquatic ecosystems requires experimental measurements of nutrient excretion and release rates. This study reports the results of an experimental assessment of nitrogen and phosphorous excretion rates by tubificid worms, which are among the dominant species in the Prahova River (Rıˆ ßs noveanu et al., 2001b). An attempt to evaluate the significance of nutrient excretion by tubificid worms was made by relating nutrient excretion rates to the biomass of tubificids in sediments and its comparison to the nutrient river water load.
Materials and methods Description of the site studied River Prahova is a second order tributary of the River Danube. Its basin has an area of 3740 km2 and is located in the Carpathian and Pontic ecoregions, between: 4445¢–4530¢ Northern latitude and 2530¢–2630¢ Eastern longitude (Fig. 1). The catchment is covering mountains, hills and plains in lower stretch from altitudes of more than 2000 m to about 100 m above the sea level. The
accelerated process of urbanization under the impact of intensive tourism, as well as developmnent of industry and intensive agriculture increased continuously the volume of wastewaters discharged into the river. An increase of organic compounds, as well as of phosphorous and nitrogen loads have been recorded. This was accompanied by decreasing of both surface and groundwater quality. Collection of experimental animals Tubificid worms were collected from the Prahova River from several sites between Azuga (955 m altitude) and Campina (200 m altitude) (Fig. 1). Along this sector the river valley is between 7 and 20 m wide, with a maximum depth of 45 cm and a water velocity of about 1.5 m s)1. The river bed is highly stony covered by a thin layer of rough sediment and/or bioderma. Bottom sediment taken by a stream-bed fauna sampler was gently washed through a 230 · 230 lm mesh screen. Animals retained with a small quantity of sediment on the screen were immediately transferred to the laboratory and kept in a constant temperature room in the dark for about 24 h at the experimental incubation temperature to be used for that sample.
Figure 1. Geographical position of the Prahova River.
123 Animals were prepared for the excretion experiment by being carefully picked up from sediments with a pair of tweezers, rinsed with distilled water and incubated without substrate for 12 h in river water at the experimental temperature for depuration. During that time, the worms almost completely emptied their guts. Bottom water temperatures in the months when worms used in the experiments were collected were: 2–3 C January, 4 C in March and 15 C in May. Time course experiment Average biomasses of 7.97 ± 2.99 mg (between 100 and 150 tubificid worms) were placed in glass vials containing 150 ml of filtered (GF/F) river water and incubated in dark, at constant temperature. The physico-chemical parameters of water were as follows: pH = 8.6, Ec = 240 lS cm)1, 0.003 lg P-PO4 ml)1, 0.366 lg N-NH4 ml)1. Fifty milliliter of water were successively taken from each bottle every 4 h for analysis, and the same quantity of filtered river water was returned to each vial in order to maintain enough water required by analytical methods. Ammonium and phosphate concentrations were determined for the water samples by colorimetric methods (Bremner, 1965; Kempers, 1974; Pym & Miham, 1976; Schneider, 1976; Fernandez et al., 1985). Sixteen hours after the start of incubation, worms were removed from the vials and faeces were further incubated for eight consecutive hours to evaluate nutrient release from faeces and the influence of microbial activity on this release. Temperature experiment To determine the effect of temperature on the phosphorus and nitrogen excretion of tubificid worms, the same experimental design was conducted in constant temperature room at different temperatures from 4 to 23 C. Five to six experimental units were used for each temperature. The temperature dependency of both ammonium and phosphate excretion was assessed using the Q10 values calculated as the ratio between excretion rates at two temperatures which differed by 10 C: Q10 ¼ RT10 =RT1 , where RT1 = excretion rate at
temperature T1 and temperature T1+10.
RT1þ10 = excretion rate at
Significance of nutrient excretion The significance of nutrient excretion by tubificid worms was evaluated by relating nutrient excretion rates obtained under the experimental conditions to the biomass of tubificids in sediments assessed during 2001–2002 and its comparison to the nutrient river water load. Abundances of worms in the river bed sediments were taken from Rıˆ ßs noveanu et al. (2001b). Nitrogen and phosphorous loads were calculated based on the nutrient concentrations in water and the corresponding volume of water column at the sampling sites. Water column volume per square meter was calculated based on the river bed profile and the dynamics of water level at the sampling sites.
Results Time course experiment The increase in nutrients content of incubated filtered river water is steeper during the first 8 h of the experiment (Figs. 2 and 3). Nitrogen and phosphorous excretions during the first 4 h of incubation are not significantly different (p > 0.05) from those recorded during the next 8 h of incubation. The amount of nutrients did not change after 16 h, when the worms were removed. The rate of nutrient excretion declines with time and become zero by 16 h. Not statistically differences (p > 0.05) were recorded during the first 8 h of incubation. This was the case for the entire temperature range. Temperature experiment Excretion rates of N and P increase significantly with temperature (Figs. 4 and 5) in a linear fashion: RNNH4 ¼ 0:0094T þ 0:0244
RPPO4 ¼ 0:0002T þ 0:0057
r2 ¼ 0:8725 r2 ¼ 0:9939
124
Excreted N-NH 4 (µ g. mg d.w. -1)
4,00 3,50 3,00
4 ˚C
2,50
7 ˚C 9 ˚C
2,00
15 ˚C
1,50
23 ˚C
1,00 0,50 0,00 0
5
10
15
20
25
Time (h)
Figure 2. The average N-NH4 amount (+SD) excreted by tubificid worms at several temperatures. Arrows indicates the moment that worms were removed from vials.
Excretion P-PO 4 (µ g.mg d.w. -1)
0,20 0,18 0,16 0,14
4 ˚C
0,12
7 ˚C
0,10
9 ˚C
0,08
15 ˚C
0,06 0,04 0,02 0,00 0
5
10 15 Time (h)
20
25
Excreted N-NH 4 (ug. mg d.w. -1 h -1)
Figure 3. The average amount (+SD) of P-PO4 excreted by tubificid worms at several temperatures. Arrow indicates the moment that worms were removed from vials.
0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 2
7
12
17
22
Temperature ( o C)
Figure 4. Temperature dependence of N-NH4 excretion rates (±SD) of tubificid worms
Excreted P-PO 4 (ug.mg d.w. -1 . h -1)
125 0,016 0,014 0,012 0,010 0,008 0,006 0,004 0,002 0,000 2
7
12
17
22
o
Temperature ( C)
Figure 5. Temperature dependence of P-PO4 excretion rates (±SD) of tubificid worms.
The Q10 for ammonium excretion appears to change between 4–15 C and 15–23 C (Fig. 4). For the lower temperature range the equation is: RNNH4 ¼ 0:0039T þ 0:0664
r2 ¼ 0:8447
The Q10 of ammonium excretion was calculated as 1.48 for the lower temperature range, while for the 4–23 C interval, it is 2.52. The value Q10 that describes the temperature dependency of phosphate excretion rate within the range 4–23 C is 1.31. Discussion In our study, nitrogen and phosphorous excretion rates were measured using river filtered water as the incubation medium, without substrate. Although this brings higher base line nutrient level and lower stability in time than would a nutrient-
free medium, it represents the natural medium of benthic invertebrates used in the experiment. In order to minimize the negative aspects related to the use of river water, we used water from the station with the lowest measured nutrient concentrations. It has been demonstrated in previous studies that excretion rates of tubificids are not influenced by the presence/absence of substrate (Gardner et al., 1983; Nalepa et al., 1983) during a 24-hour incubation. Although food deprivation affects the metabolism of these invertebrates, excretion rate seems to be a less sensitive parameter than other metabolic indicators, such as respiration (Nalepa et al., 1983). We measured ammonium excretion and not total inorganic N excretion, as ammonium is thought to be the major inorganic N form excreted by tubificids (Fukuhara & Sakamoto, 1987). Ammonium excretion rates reported in literature vary by up to one order of magnitude (Table 1).
Table 1. Nutrient (N-NH4 and P-PO4) excretion rates reported by several authors for tubificid worms in different experimental conditions Reference
T (C)
Excretion rate
Reference
T (C)
(lg N mg d.w.)1 h)1) Gardner et al. (1983)
10
0.042–0.073
22
0.1512
Fukuhara & Sakamoto (1987) Fukuhara & Yasuda (1989)
20 15
Rıˆ ßs noveanu et al. (2001a) Present study
Excretion rate (lg P mg d.w.)1 h)1)
Nalepa et al. (1983)
16
0.004–0.005
0.071 0.0246
Fukuhara & Yasuda (1985) Fukuhara & Sakamoto (1987)
15 20
0.002 0.003
23
0.155
Rıˆ ßs noveanu et al. (2001a)
23
0.006
4
0.076
Present study
23
0.266
4
0.007
23
0.010
126 Our rates are higher than those of other authors. This variation may be related to experimental conditions, characteristics of individual species and/or their environment. To our knowledge, this is for the first time that tubificids from streams were used to investigate excretion process. As differences between excretion rates for 0– 4 h interval are not statistically significant from those for 0–8 h interval, a constant excretion rate can be calculated for up to 8 h. The decrease of ammonium release rates after the first 8 h was also observed by other authors (Fukuhara & Yasuda, 1985, 1989; Rıˆ ßs noveanu et al., 2001a) and can be due to the emptying of guts. The amount of ammonium did not change after the removal of animals (at 16 h) and this suggests that excretion rates were not significantly affected by nutrient release from faeces. Rates of phosphate release are almost constant for about 8 h. This was also found by other authors, who obtained relatively constant rates for the first 10 h (Fukuhara & Yasuda, 1985, 1989; Rıˆ ßs noveanu et al., 2001a). Generally, the variation of phosphate excretion rates, as reported in literature, is much smaller compared to that for ammonium (Table 1). With the exception of one value recorded at 23 C, rates we obtained are only slightly higher than those in literature. Generally, the metabolic rates of poikilotherm organisms show an increase with temperature. Although a linear dependency of ammonium excretion rate was obtained, a steeper increase at 23 C was found. This could be the result of abnormal physiological behavior of organisms under a higher temperature than the normal range in their environment. A similar trend was found for phosphate. In this case, there is no significant increase in excretion rates within the temperature interval of 4–9 C, and this can be explained by worms adaptation to a relatively wide daily temperature range in the region studied. Usually Q10 values between 2 and 3 are characteristic for invertebrates (Prosser, 1973). The lower value for ammonium excretion rates in the lower temperature range (4–15 C) and for phosphate excretion shows a smaller sensitivity to temperature increase. Tubificid worms excrete nutrients through nephridia located in each segment of their body.
Therefore, nutrients are released both into the water column and sediments. From sediments, nutrients may reach the water column by molecular diffusion or through channels created from bioturbation. The significance of nutrients excretion into stream nutrient pools can be put into perspective by comparing its contribution to the nutrient water column load. Based on the tubificid abundance in these sites of the Prahova River (Rıˆ ßs noveanu et al., 2001b), if we presume that all excreta eventually enters the water column, then we can calculate that these invertebrates add 39.17 mg N m)2 day)1 and 0.49 mg P m)2 day)1. These values accounts for 17.16 and 7.56% of the nutrient load in the river water, respectively. This suggests the importance the tubificid worms may have in the nutrient cycling in rivers. More studies are needed to better clarify this role.
Acknowledgements The National Council of Scientific Research in Higher Education System provided financial support for this research in Higher Education System. The authors would like to thank Dr Kate Lajtha for English revision of the manuscript. Thanks are also due to our technician Botos Zamfira for her aid in the chemical analyses and to our Masters students for their help in the preparation of worm for the experiments.
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