Water Research Pergamon Press 1969. Vol. 3, pp. 367-373. Printed in Great Britain

E S T I M A T E S O F P E R I P H Y T O N M A S S A N D S T R E A M

B O T T O M A R E A U S I N G P H O S P H O R O U S - 3 2

D. J. NELSON, N. R. KEWRN*, J. L. WILHM t and N. A. GlUvvrrtt

Radiation Ecology Section, Health Physics Division Oak Ridge National Laboratory, Oak Ridge, Tennessee, U.S.A.

(Received 9 January 1969)

Abstract--The standing crop of periphyton and actual surface area in a 100m section of a small stream were estimated by applying material balance techniques to an experimental s2p release. About 75 per cent of the introduced 32p was retained initially by periphyton in the study section. Based on the quantity of 32p in periphyton samples, per nag and per cm 2, the total standing crop of periphyton and the bottom area of the section were estimated as 1.5 kg and 560 m 2, respectively.

INTROD UCTION

K N O ~ E of the behavior of a radionuclide in the environment can be used to obtain ecological data that are dit~cult or impractical to obtain by conventional methods. Because of the difficulty in making measurements, only limited data are available concerning the standing crop of periphyton (Aufwuchs) and the actual surface of stream bottoms. The periphyton includes all organisms attached to the substrate and these organisms are important in streams because they constitute the base of the food chain. This paper describes a rapid method for determining the biomass of periphyton and the bottom surface area of a small, rocky stream using 32p.

Previous research with ~2p in aquatic environments showed that it was accumulated rapidly by phytoplankton. RmL~ 0956) reported that 95 per cent of the 32p added to lake water was taken up by phytoplankton within 20 minutes. Direct uptake of S2p occurred primarily among the phytoplankton and periphyton, and 32p movement to higher levels in the food chain appeared to be chiefly by means of ingestion (DAvis and FOSTEg, 1958; FOSTER, 1963). COFFIN et al. 0949) did not detect 32p in fish in a small lake until 50 hours after its introduction.

Previous surveys of the behaviour of 32p introduced into White Oak Creek, Ten- nessee, were conducted during July 1962, 1963, and 1965. The following conclusions regarding translocation of phosphorus were noted:

(1) as much as 95 per cent of the introduced 32p was retained within the study- reach;

(2) periphyton rapidly accumulated large quantities; (3) small concentrations occurred in inorganic sediments;

* Present Address: Department of Fisheries and Wildlife, Michigan State University, East Lansing, Michigan, U.S.A. I" U.S. Atomic Energy CommiAsion Postdoctoral Fellow under appointment from the Oak Ridge

Associated Universities. Present address: Department of Zoology, Oklahoma State University, Still- water, Oklahoma 74074, U.S.A.

367

368 D.J. NELSON, N. R. KEVERN, J. L. WILrrM and N. A. G~nnTH

(4) accumulation by consumer organisms such as snails, odonates, hellgramites, crayfish, fish, and salamanders was low initially and increased gradually for several days.

The results are in agreement with published information on the behavior of 32p in a stream environment (BALL and HOOPER, 1963). Because of this consistent behavior, material balance techniques may be used to determine the amount of released 32p that is retained in a study-reach of a stream. This method is indirect but is considered reasonably accurate since 32p initially is accumulated by periphyton. Also, the periphyton in the creek community would contain the most biomass with much lesser quantities in consumer organisms. Because of the concentration of a2p in periphyton and the relatively large biomass, errors introduced by uptake in other components of the community would be minor. Furthermore, with data on the 32p activity per unit area the actual surface area of the stream bottom can be estimated. At the present there is no convenient method of measuring the actual surface area of an irregular stream bottom.

METHODS

A 100-m section of White Oak Creek was divided into five 20-m subsections, each with at least one riffle and pool. These subsections were used for the sampling of periphyton on glass slides and rocks. The creek bottom was covered predominantly with gravel-rubble size, ~ l a r - s h a p e d rocks and smaller areas of compact clay or sand-silt mixtures. Low water levels occurred during the study, and no scouring dis- charges had occurred for at least 5 weeks. Hence, a relatively uniform growth of periphyton covered the creek bottom. Aquatic macrophytes were rare. As a prelimin- ary precaution, tree branches, roots, grass and other herbaceous vegetation dangling in the creek were removed.

The equipment used to introduce the 32p consisted of a 4-1. polyethylene bottle, a funnel, and a section of glass tubing drawn to a fine tip and attached with rubber tubing to the funnel. The bottle containing the isotope solution was inverted over the funnel, and the entire apparatus was supported on a ring stand placed in the middle of the creek. Empirical testing of the apparatus showed that a 30-rain release was obtained by diluting the 32p solution to 3650 ml.

Creek discharge was measured at a weir located about 30 m downstream from the study-reach. Measured discharge at the time of the radionuclide release was 8.7 x l06 ml per 30 rain. The maximum permissible concentration of 32p in water for occupa- tional exposure (40-hr week) is 5 x 10-* #Ci/ml (ICRP, 1959), Accordingly, 4.35 mCi of 32p released during a 30-min period was permissible. The actual release was 4.3 mCi (9.5 x 109 dis/rain, of P(V) in 0.4 N HC1), giving a maximum 32p con- centration, assuming instantaneous dilution, of 1.1 x l0 s dis/min/ml of creek water. The amount of 32p released was directly proportional to the isotope release period, While the release period was arbitrary, previous experience showed that 30 rain was sufficiently long to provide an adequate tag for the periphyton. The 32p was introduced at the head of the test section.

The quantity of S2p retained in the stream section was determined by measuring the activity in the water at the end of the 100-m study-reach and comparing this value with the activity introduced at the upstream station. Water masses to be sampled at the end

Periphyton Mass and Phmphorus-32 369

of the study section were traced by introducing fluorescein dye at the upstream station one minute before initiating the 32p release. A background sample was taken prior to the appearance of the dye. The second sample was collected as the initial dye passed, and the next samples were taken 2 and 5 rain later with subsequent samples taken at 5-rain intervals. Although the release lasted 30 rain, downstream sampling continued for 100 rain because of longitudinal dispersion. Water samples were collected in 210-ml polyethylene bottles. Each of the 22 samples was taken from the same point in the stream.

Activity sorbed by particulate matter and remaining dissolved in water were determined by filtering 200 ml of each sample through a 0.45/l membrane filter. The filter flask was rinsed with 10 ml 0.1 S HC1 and the filter removed and allowed to dry in a desiccator for at least 24 hr before being weighed, placed on a planchet and counted. A 4-ml aliquot of filtered water was transferred to a planchet, spread with a wetting agent, and dried before counting. Activity of both the particulate and dis- solved matter was expressed in dis/rain/1. Corrections were made for background, counter efficiency, radioactive decay, and acid volume. Total activity passing the down- stream station was calculated by multiplying mean activity of particulate and dis- solved matter by total discharge during the sampling period.

The material balance for 32p in the study-reach was determined from the water samples. During the initial hours after the 32p release, most of the activity was incor- porated in the periphyton. Then the following relationships existed:

P~-P~ ffi P, (I)

where Ps = activity introduced in spike or at an upstream station, Pa -- activity at the point 100 m downstream, and P, -- activity retained in section. All activities are expressed as dis/rain. Further

P, (dis/rain) = standing crop ofperiphyton in stream section (rag), (2)

Pw (dis/rain/rag)

where Pw is the activity/unit wt. in periphyton as determined on natural or artificial substrates. Likewise,

Pr (dis/rain) total bottom area (cm2), where Po is Po (dis/min/cm 2) = activity/unit area ofperiphyton. (3)

Uptake of 32p by periphyton was determined by collecting samples from both artificial and natural substrates 4 hr after introduction of the 32p. Each artificial substrate consisted of a glass slide, with an exposure area of 45 cm 2, attached with waterproof tape to the side of a 40-penny nail. The nail supporting the slide was driven into the stream bottom so the slide was held vertically and oriented parallel with stream flow. Five slides were suspended in each riflte and pool of each subsection 35 days prior to releasing 32p. Slides were carried to the laboratory and periphyton was scraped from the slides into tared planchets. Activity on natural substrates was determined by collecting five rocks from each riffle and pool from the uppermost and lowermost subsections. The rocks were returned to the laboratory in plastic bags and samples ofperiphyton were scraped onto tared planchets. Activity of periphyton from both types of substrates was determined as dis/rain/rag ash-free weight and dis/rain/era 2 of exposed surface.

F W

370 D.J. NELSON, N. R. KEVERN, J. L. WILHM and N. A. GRIFFITH

Activity on inorganic sediments and leaves was determined to evaluate uptake by other substances. Prior to the release of 32p, inorganic sediments in several areas were exposed by removing overlying periphyton. Four hours after initiation of the 32p release, a glass tube and squeeze bulb were used to aspirate sediment samples. A 4-ml aliquot of the water-sediment mixture was placed on a tared planchet for counting and its activity was expressed in dis/min/mg dry wt. Fallen leaves were collected at the same time as inorganic sediments. A disk 3.2 cm in diameter was cut from each leaf, dried, glued onto a planchet, and radioassayed.

Previous experiments had shown sorption of 32p occurred primarily at the surface of substrates. Data obtained from leaves were expressed conveniently as dis/min/cm 2. Data from sediments could not be expressed so simply but 32p activity obtained from the aspirated surficial layer was adequate for comparative purposes.

RESULTS

Activity retained in study section The initial volume of water containing fluorescein dye required 17 min to flow

through the 100-m study section. The background sample had a concentration of 1.1 x 103 dis/min/l. A plot of 32p concentrations in successive water samples produced a bell-shaped curve with a peak of 2.9 x 10 s dis/rain/1 in the water c.~llected 30 rain after appearance of the dye. The dis/min/1 of the 70-min water sample decreased to 3.7 x 10 a, and the 90-rain collection approximated background.

Stream discharge was 2.9 x 10 2 l/rain, and mean dis/min/1 of dissolved and particul- ate matter was 8.0 x 104 and 3.1 x l0 a, respectively. Total activity in the effluent from the downstream station was the product of mean activity and discharge during the 100-min sampling period. Thus, (8.3 x 104 dis/rain/1 (2.9 x 104 l.) = 2.4 x 109 dis/rain. Activity of 32p introduced at the upstream station was 9.5 x 109 dis/rain and activity leaving the study area was 2.4 x 10 9. By subtraction (1), 7.1 x 109 dis/rain, or about 75 per cent of the introduced a2p, was retained within the study section.

Activity on sediments and leaves The 32p concentration (all data are the mean 4- one standard error) in nine sediment

samples was 8.54-5.9 dis/min/mg and in 9 leaf samples was 103:t:44 dis/rain/era 2. Penphyton on artificial substrata sorbed 254 times as many dis/rain/rag as sediments and 48 times more dis/min/cm 2 than leaves. High activity on three leaves which had been in the creek longer and had accumulated penphyton growths accounted for a larger mean 32p concentration. In cases when a few values are extreme, the median may be a better measure of the central tendency. Using median activity on leaves (31 dis/min/cm 2) for comparison, periphyton on artificial substrata sorbed 157 times as many dis/min/cm 2. These data show comparatively little activity was sorbed by sediments and fresh leaves.

Periphyton on artificial and natural substrates The mean ash-free weight of periphyton on artificial substrata in mg/cm 2 was

0.275_+0,045 in riffles and 0.3334-0.025 in pools and with a mean on all 50 slides of 0.304 4- 0.026 mg/cm 2. Lower values in riffles probably resulted from slower accumula- tion of perphyton on slides and greater sloughing. DUNCAN'S (1955) new multiple range test revealed that riffles in subsections 2-5 were significantly different (P = 0.01)

Periphyton Mass and Phosphorus-32 371

from subsection 1, but not from each other. Higher weights in the riffle of subsection 1 probably resulted from less shading. Means in pools of the five subsections were not significantly different from each other. A t-test showed that no significant differences (P = 0.01) existed between means of pools and riffles. Thus, the grand mean of pools and riffles was used in subsequent calculations.

The mean weight of periphyton on 20 rocks was 4.95 + 0.696 mg/cm 2, a statistically different value from that obtained on artificial substrates. These rocks were selected because they had flat surfaces that could be measured easily and the flat surfaces were also horizontal surfaces. Horizontal surfaces accumulate 6-12 times as much peri- phyton as vertical surfaces (CAsTI~NHOLZ, 1961). In this instance we introduced a bias by selecting flat rocks which were atypical of the stream bed materials with respect to type and orientation. Because of this bias, data obtained from rocks were not included in calculations of periphyton biomass.

Estimation of standing crop Phosphorus-32 concentrations of periphyton on both artificial and natural sub-

strates in riffes exceeded that in pools. Mean activity of periphyton in dis/min/mg on slides was 6800-+ 560 in riffes and 3000-+260 in pools, while activity on the rock film was 800-+270 in riffles and 280+60 in pools. WHrrFom) and SCHOMACrmR (1964) studied effects of current on 32p uptake in several species of lotic and lentic algae and reported that all species studied had higher rates of uptake in a current. Lotie species showed a greater response to a current, indicating a higher metabolic rate.

Although asia-free weight of periphyton on rocks exceeded that on slides, dis/min/mg of periphyton was greater on slides. Uptake of 32p appeared to be a surface pheno- menon because of the availability of the 32p to surface layers. Hence, thicker peri- phyton growths on rocks contained more underlying areas not sorbing 32p. Periphyton on 50 slides in riffles and pools throughout the stream section contained a mean con- centration of 4900+470 dis/min/mg. Since most activity was accumulated in peri- phyton, (2) was used to estimate the total standing crop of periphyton in the 100-m section:

7.1 x 109 dis/min = 1 .5 x 106 mg.

4.9 x 103 dis/min/mg

Estimation of bottom area Periphyton on artificial substrates in pools contained a mean concentration of

1000+145 dis/min/cm 2 and 32p activity on substrates in riffes was 1500_+230 dis/min/cm 2. As with the data for dis/min/mg, riffle values exceeded those of pools. However, activity per unit area on artificial substrates approximated values for rocks which were 1300 4-150 dis/min/cm 2 in pools and 200 ± 550 dis/rain/era 2 in riffles, again suggesting that uptake was largely a surface phenomenon. The mean a2p concentra- tion on all slides was 1260± 140 dis/rain/era 2. Since most activity was concentrated in periphyton, and since most bottom surfaces were coated with periphyton, the ratio between dpm retained in the section and mean dis/min/cm 2 of periphyton should approximate the cm 2 of bottom area. Using (3)total surface area of the stream bottom is

7.1 x 109 dis/rain 1.26 x 103 dis/min/cm 2 = 5.6 x 106 cm 2.

372 D.J. NELSON, N. R. KEVERN, J. L. WILHM and N. A. GRIFFITH

Riffles in the study section were 59.0 m long with a mean width of 1.15 m, while pools were 41 m long with a mean width of 1.61 m. Bottom areas, as calculated from surface measurements, were 68 m 2 for riffles and 66 m 2 for pools. The similarity of these values justified combining data from riffle and pool periphyton samples and using one mean for calculating bot tom area of the entire stream.

Bottom area calculated from surface measurements was 134 m z, while total bot tom area estimated by 32p uptake was 560 m 2. The latter estimate exceeds the former by a factor of four. In view of the irregular topography of the stream bottom, the value obtained from uptake data is a better estimation of total bot tom area than surface measurements of length and width.

Standing crop expressed on a total area basis was 2.7 g/m 2 but 11.2 g/m 2 using surface measurements. The first value is more realistic since it was based on the area actually available for periphyton attachment. Both expressions of standing crop and the standing crop as measured on artificial substrates were compared with estimates f rom other streams (TABLE 1). Estimates reported on the same weight basis and on an actual areal basis can be compared directly. The actual area is available when fiat surfaces were used, either slides or rocks, or when estimated as was done in this study. The higher estimates in TABLE 1 apparently represented unusual growth conditions or were reported on a surface area that contained considerably more actual area.

TABLE 1. COMPARISON OF STANDING CROPS OF PERIPHYTON IN VARIOUS STREAMS

Source Locality Remarks Biomass (g/m 2) Dry wt. Ash-free wt

McCoN~SLL and SIGL~a (1959) Logan R., Utah

McI~rrn~ et aL (1964) Artificial lab streams

KOeAYASI (1961) Arakawa R., Japan CusmNG (1966) Columbia R., Wash. DRUM (1963) Des Moines R., Iowa KEVEaN et aL Artificial lab streams

(1966) Spring in Tennessee Present study White Oak Creek,

Tenn.

Rocks within m 2 25

Rocks in measured area 187 Rock surface (25 cm 2) 2.5-7.0 Glass slides (14)* 4.2 1.8 Flat rock surfaces 210 Plexiglas slides (34)* 12 Plexiglas slides (35)* 5.2 Glass slides (35)* 15.6 3.0 Area, estimated (560 m 2) 2.7 Area, linear measurements (134 m 2) 11.2

* Exposure time in days.

The technique is adaptable to shallow streams where periphyton is a major com- ponent of the biota. Selection of the upstream release site to obtain a known dis- persion pattern is important. Knowledge of both lateral and longitudinal dispersion of introduced materials can be obtained with dyes. An extended sampling period at the downstream station will compensate for longitudinal dispention. In the narrow stream we studied, a prompt lateral dispersion was effected by releasing the 32p in an area where thorough mixing occurred in a small rapids.

Application of these methods should not be considered restrictive to 32p and periphyton. Any combination of a radionuclide and a specific receptor surface would be suitable. This method appears to be particularly useful for determining surface

Periphyton Mass and Phosphorus-32 373

areas o f uneven s t ream channels , and as such might prove appl icab le for de te rmining hydrau l ic pa ramete r s such as bed roughness.

Acknawledgements---Research sponsored by the U.S. Atomic Energy Cornmi~ion under contract with the Union Carbide Corporation. We should like to acknowledge the preliminary information on the behavior of s2p in White Oak Creek obtained in field exercises completed by the participants in the Oak Ridge Institute of Nuclear Studies Institutes of Radioecology during 1962, 1963, and 1965.

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