Nitrogen and Phosphorous Excretion Rates by Tubificids from the Prahova

Hydrobiologia (2006) 553:121–127 Springer 2006 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 ßsnoveanu & 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 )1 )1 )1 )1 N mg d.w. h and 0.0065–0.01 lg P mg d.w. h , 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 Nitrogen and phosphorous exchange at the water–sediment interface is controlled by many The Danube is the second largest river in Europe. complex physico-chemical factors, as well as by It has a catchment area of 807,000 km2 that biological processes (Bostrom et al., 1982). Zoo- stretches over 13 states. Eutrophication of the benthos can inﬂuence nutrient dynamics at the Danube Basin is a key environmental concern that water–sediment interface through excretion of has emerged in the last few decades, as scientists nutrient compounds, and through continuous and managers have observed sharp increases in release of nutrients from sediments through nitrogen and phosphorous concentrations in channels created by bioturbation activity (Fuku- freshwater lakes and rivers throughout the region. hara & Yasuda, 1985, 1989; Fukuhara & Saka- A management plan for the preservation of good moto, 1987, 1988; Rıˆ ßsnoveanu et al., 2002, 2004)). water quality is critically needed, but any such There is relatively little known about the eﬀects of plan must be based on detailed knowledge of macroinvertebrates on nutrient mineralization and nutrient emission sources, pathways and sinks in exchange processes during bioturbation, particu- the catchment area. larly in stream ecosystems. 122

The assessment of the role of macroinverte- accelerated process of urbanization under the im- brates in nutrient cycling in aquatic ecosystems pact of intensive tourism, as well as developmnent requires experimental measurements of nutrient of industry and intensive agriculture increased excretion and release rates. This study reports the continuously the volume of wastewaters dis- results of an experimental assessment of nitrogen charged into the river. An increase of organic and phosphorous excretion rates by tubificid compounds, as well as of phosphorous and nitro- worms, which are among the dominant species in gen loads have been recorded. This was accom- the Prahova River (Rıˆ ßsnoveanu et al., 2001b). An panied by decreasing of both surface and attempt to evaluate the significance of nutrient groundwater quality. excretion by tubificid worms was made by relating nutrient excretion rates to the biomass of tubificids Collection of experimental animals in sediments and its comparison to the nutrient river water load. 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 Materials and methods 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 Description of the site studied highly stony covered by a thin layer of rough sediment and/or bioderma. River Prahova is a second order tributary of the Bottom sediment taken by a stream-bed fauna River Danube. Its basin has an area of 3740 km2 sampler was gently washed through a and is located in the Carpathian and Pontic eco- 230 · 230 lm mesh screen. Animals retained with a regions, between: 4445¢–4530¢ Northern latitude small quantity of sediment on the screen were and 2530¢–2630¢ Eastern longitude (Fig. 1). The immediately transferred to the laboratory and kept catchment is covering mountains, hills and plains in a constant temperature room in the dark for in lower stretch from altitudes of more than about 24 h at the experimental incubation tem- 2000 m to about 100 m above the sea level. The perature to be used for that sample.

Figure 1. Geographical position of the Prahova River. 123

Animals were prepared for the excretion temperature T1 and RT1þ10 = excretion rate at experiment by being carefully picked up from temperature T1+10. sediments with a pair of tweezers, rinsed with distilled water and incubated without substrate for Significance of nutrient excretion 12 h in river water at the experimental temperature for depuration. During that time, the worms The significance of nutrient excretion by tubificid almost completely emptied their guts. worms was evaluated by relating nutrient excre- Bottom water temperatures in the months tion rates obtained under the experimental condi- when worms used in the experiments were col- tions to the biomass of tubificids in sediments lected were: 2–3 C January, 4 C in March and assessed during 2001–2002 and its comparison to 15 C in May. the nutrient river water load. Abundances of worms in the river bed sedi- Time course experiment ments were taken from Rıˆ ßsnoveanu et al. (2001b). Nitrogen and phosphorous loads were calcu- Average biomasses of 7.97 ± 2.99 mg (between lated based on the nutrient concentrations in water 100 and 150 tubificid worms) were placed in glass and the corresponding volume of water column at vials containing 150 ml of filtered (GF/F) river the sampling sites. Water column volume per water and incubated in dark, at constant temper- square meter was calculated based on the river bed ature. The physico-chemical parameters of water profile and the dynamics of water level at the were as follows: pH = 8.6, Ec = 240 lScm)1, sampling sites. )1 )1 0.003 lg P-PO4 ml , 0.366 lg N-NH4 ml . Fifty milliliter of water were successively taken from each bottle every 4 h for analysis, and the same Results quantity of filtered river water was returned to each vial in order to maintain enough water Time course experiment required by analytical methods. Ammonium and phosphate concentrations were determined for the The increase in nutrients content of incubated fil- water samples by colorimetric methods (Bremner, tered river water is steeper during the first 8 h of 1965; Kempers, 1974; Pym & Miham, 1976; the experiment (Figs. 2 and 3). Nitrogen and Schneider, 1976; Fernandez et al., 1985). Sixteen phosphorous excretions during the first 4 h of hours after the start of incubation, worms were incubation are not significantly different (p > 0.05) removed from the vials and faeces were further from those recorded during the next 8 h of incu- incubated for eight consecutive hours to evaluate bation. The amount of nutrients did not change nutrient release from faeces and the influence of after 16 h, when the worms were removed. microbial activity on this release. The rate of nutrient excretion declines with time and become zero by 16 h. Not statistically Temperature experiment differences (p > 0.05) were recorded during the first 8 h of incubation. This was the case for the To determine the effect of temperature on the entire temperature range. phosphorus and nitrogen excretion of tubificid worms, the same experimental design was con- Temperature experiment ducted in constant temperature room at different temperatures from 4 to 23 C. Five to six experi- Excretion rates of N and P increase significantly mental units were used for each temperature. The with temperature (Figs. 4 and 5) in a linear fashion: temperature dependency of both ammonium and 2 phosphate excretion was assessed using the Q10 RNNH4 ¼ 0:0094T þ 0:0244 r ¼ 0:8725 values calculated as the ratio between excretion rates at two temperatures which differed by 10 C: R 0:0002T 0:0057 r2 0:9939 Q10 ¼ RT10 =RT1 , where RT1 = excretion rate at PPO4 ¼ þ ¼ 124

4,00

) 3,50 -1

3,00 4 ˚C mg d.w. . 2,50 7 ˚C g µ

9 ˚C (

4 2,00 15 ˚C 1,50 23 ˚C

1,00

Excreted N-NH Excreted 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.

0,20 )

-1 0,18 0,16 0,14 mg d.w.

. 4 ˚C g 0,12

µ 7 ˚C ( 4 0,10 9 ˚C 0,08 15 ˚C 0,06 0,04 0,02 Excretion P-PO Excretion 0,00 0 5 10 15 20 25 Time (h)

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 -1 h -1 0,30

0,25 mg d.w. . 0,20 (ug 4 0,15

0,10

0,05

Excreted N-NH Excreted 0,00 2 7 12 17 22 Temperature ( oC)

Figure 4. Temperature dependence of N-NH4 excretion rates (±SD) of tubiﬁcid worms 125

) 0,016 -1

h 0,014 -1 . 0,012

0,010 mg d.w. .

(ug 0,008 4 0,006

0,004 0,002

Excreted P-PO Excreted 0,000 2 7 12 17 22 Temperature ( oC)

Figure 5. Temperature dependence of P-PO4 excretion rates (±SD) of tubiﬁcid worms.

free medium, it represents the natural medium of The Q10 for ammonium excretion appears to change between 4–15 C and 15–23 C (Fig. 4). benthic invertebrates used in the experiment. In For the lower temperature range the equation is: order to minimize the negative aspects related to the use of river water, we used water from the 2 RNNH4 ¼ 0:0039T þ 0:0664 r ¼ 0:8447 station with the lowest measured nutrient concentrations. It has been demonstrated in previous The Q10 of ammonium excretion was calculated as 1.48 for the lower temperature range, while for the studies that excretion rates of tubificids are not influenced by the presence/absence of substrate 4–23 C interval, it is 2.52. The value Q10 that describes the temperature dependency of phos- (Gardner et al., 1983; Nalepa et al., 1983) during a 24-hour incubation. Although food deprivation phate excretion rate within the range 4–23 Cis 1.31. affects the metabolism of these invertebrates, excretion rate seems to be a less sensitive parame- ter than other metabolic indicators, such as respi- Discussion ration (Nalepa et al., 1983). We measured ammonium excretion and not In our study, nitrogen and phosphorous excretion total inorganic N excretion, as ammonium is rates were measured using river filtered water as thought to be the major inorganic N form excreted the incubation medium, without substrate. by tubificids (Fukuhara & Sakamoto, 1987). Although this brings higher base line nutrient level Ammonium excretion rates reported in literature and lower stability in time than would a nutrient- 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) Excretion rate (lg N mg d.w.)1 h)1) (lg P mg d.w.)1 h)1)

Gardner et al. (1983) 10 0.042–0.073 Nalepa et al. (1983) 16 0.004–0.005 22 0.1512 Fukuhara & Sakamoto (1987) 20 0.071 Fukuhara & Yasuda (1985) 15 0.002 Fukuhara & Yasuda (1989) 15 0.0246 Fukuhara & Sakamoto (1987) 20 0.003 Rıˆ ßsnoveanu et al. (2001a) 23 0.155 Rıˆ ßsnoveanu et al. (2001a) 23 0.006 Present study 4 0.076 Present study 4 0.007 23 0.266 23 0.010 126

Our rates are higher than those of other authors. Therefore, nutrients are released both into the This variation may be related to experimental water column and sediments. From sediments, conditions, characteristics of individual species nutrients may reach the water column by and/or their environment. To our knowledge, this molecular diffusion or through channels created is for the first time that tubificids from streams from bioturbation. The significance of nutrients were used to investigate excretion process. excretion into stream nutrient pools can be put As differences between excretion rates for 0– into perspective by comparing its contribution to 4 h interval are not statistically significant from the nutrient water column load. Based on the those for 0–8 h interval, a constant excretion tubificid abundance in these sites of the Prahova rate can be calculated for up to 8 h. The de- River (Rıˆ ßsnoveanu et al., 2001b), if we presume crease of ammonium release rates after the first that all excreta eventually enters the water col- 8 h was also observed by other authors umn, then we can calculate that these inverte- (Fukuhara & Yasuda, 1985, 1989; Rıˆ ßsnoveanu brates add 39.17 mg N m)2 day)1 and 0.49 mg et al., 2001a) and can be due to the emptying Pm)2 day)1. These values accounts for 17.16 of guts. The amount of ammonium did not and 7.56% of the nutrient load in the river change after the removal of animals (at 16 h) water, respectively. This suggests the importance and this suggests that excretion rates were not the tubificid worms may have in the nutrient significantly affected by nutrient release from cycling in rivers. More studies are needed to faeces. better clarify this role. Rates of phosphate release are almost constant for about 8 h. This was also found by other authors, who obtained relatively constant rates for Acknowledgements the first 10 h (Fukuhara & Yasuda, 1985, 1989; Rıˆ ßsnoveanu et al., 2001a). Generally, the variation The National Council of Scientific Research in of phosphate excretion rates, as reported in liter- Higher Education System provided financial sup- ature, is much smaller compared to that for port for this research in Higher Education System. ammonium (Table 1). With the exception of one The authors would like to thank Dr Kate Lajtha value recorded at 23 C, rates we obtained are for English revision of the manuscript. Thanks are only slightly higher than those in literature. also due to our technician Botos Zamfira for her Generally, the metabolic rates of poikilotherm aid in the chemical analyses and to our Masters organisms show an increase with temperature. students for their help in the preparation of worm Although a linear dependency of ammonium for the experiments. 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 References range in their environment. A similar trend was Bostrom, B., M. Jansson & C. Forsberg, 1982. Phosphorus found for phosphate. In this case, there is no release from lake sediments. Archiv fu¨ r Hydrobiolgie 18: significant increase in excretion rates within the 5–59. temperature interval of 4–9 C, and this can be Bremner, J. M., 1965. Inorganic Forms of Nitrogen. In Black, C. A. explained by worms adaptation to a relatively et al., (eds.), Methods of Soil Analysis. American Society of wide daily temperature range in the region stud- Argon Inc., Madison, WI: 1179–1237. Fernandez, J. A., F. X. Niell & J. Lucerna, 1985. A rapid and ied. Usually Q10 values between 2 and 3 are sensitive automated determination of phosphate in natural characteristic for invertebrates (Prosser, 1973). waters. Limnology and Oceanography 30: 227–230. The lower value for ammonium excretion rates in Fukuhara, H. & K. Yasuda, 1989. Ammonium excretion by the lower temperature range (4–15 C) and for some freshwater zoobenthos from an eutrophic lake. Hyd- phosphate excretion shows a smaller sensitivity to robiologia 173: 1–8. Fukuhara, H. & M. Sakamoto, 1987. Enhancement of inor- temperature increase. ganic nitrogen and phosphate release from lake sediment Tubificid worms excrete nutrients through by tubificid worms and chironomid larvae. Oikos 48: nephridia located in each segment of their body. 312–320. 127

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