Evaluation of a Bio-Organic Catalyst
in Channel Catfish, Ictalurus punctatus,
Ponds
Julio F. Q ueiroz
( 0
^Claude E. Boyd
A m it Gross
A B S T R A C T . A bio-organic catalyst w as tested in ponds used to grow channel catfish, Ictalurus pu n cta tu s, at A uburn, A labam a, for its effect on w ater quality, soil organic carbon, and channel catfish production. A lthough there w ere no significant differences (P > 0 .1 ), ponds treated w ith the bio-organic catalyst tended to have higher concentrations o f dissolved oxygen than control ponds during su m m er m onths even though all ponds w ere aerated m echanically. D ata on w ater quality and soil organic carbon suggested that the bio-or ganic catalyst caused a slight but statistically insignificant inhibition o f phytoplankton productivity, w hich in turn lessened the nighttim e oxygen dem and. A lthough fish production did not increase as the result o f g reater dissolved oxygen availability, fish survival w as h igher in the treated ponds (P = 0.1). A t the m axim um daily feeding rate o f 75 kg/ha, w ater quality w as not severely im paired in any o f the ponds. T he b io-organic catalyst p ro d u ct m ig h t have greater b enefits in p o n d s w ith h ig h er stocking and feeding rates. In fo rm a tion on the m ech an ism o f action o f bio-catalyst ad d itio n s in pond ecosystem s w ould be useful in determ ining their potential benefits to pond aquaculture. [Article copies available fo r a fe e from The Haworth
Document Delivery Service: 1-800-342-9678. E-mail address: getinfo@ haworthpressinc. com]
Julio F. Queiroz, Claude E. Boyd, and Amit Gross, Department of Fisheries and Allied Aquacultures, Auburn University, Auburn, AL 36849.
Address correspondence to Claude E. Boyd at the above address. Journal of Applied Aquaculture, Vol. 8(2) 1998
INTRODUCTION
Substances high in enzym atic activity, such as bio-organic catalysts (B O C s), have been developed to accelerate natural m icrobial process in soil, w ater, or other m edia. T hese B O C products have been used in agri culture, w astew ater treatm ent, clean-up o f oil spills, and rem ediation o f sites contam inated w ith hazardous w astes (M etting 1992). Enzym es are catalysts that accelerate biochem ical reactions, and m icroorganism s ex crete extracellular enzym es th at are im portant in the initial stages o f d e com position. E xtracellular enzym es break large m olecules into sm aller fragm ents that can be absorbed by m icrobial cells (A nderson 1987), and they can cause transform ations o f various com pounds to accelerate m icro bial processes (D ick and T abatabai 1992). E nough success has been ob tained w ith som e o f these products to encourage com panies to produce and m arket them for a v ariety o f applications (G lass 1992). Some products purportedly im prove w ater quality, and these have been recom m ended for use in pond aquaculture. M anufacturers claim their product can im prove w ater quality through increasing the solubility o f hydrophobic m aterial, im proving gas diffusion in ponds b y increasing dissolved oxygen levels, and accelerating breakdow n o f organic m atter.
The price o f channel catfish, Icta lu ru s pu n cta tu s, has not increased appreciably over the past decade, and this has lead to greater stocking and feeding rates to increase production p er u nit area. W ater quality problem s increase as production rises, and som e farm ers are interested in the possi bilities o f using bio-organic catalysts to im prove w ater quality. Because little inform ation is available on the use o f these substances in aquaculture, the present study w as initiated to evaluate the use in channel catfish ponds o f a B O C , w hich contains derived catalysts, surface m odifying synthetic com pounds, and other proprietary ingredients.
MATERIALS AND METHODS
Ponds
N ine 400-m 2 levee ponds on the A uburn U niversity Fisheries Research U nit (FRU ), A uburn, A labam a, w ere used in this study. Ponds were square w ith earthen bottom s that gradually sloped from depths o f about 20-40 cm at the vertical edges supported b y w ooden or concrete w alls to 130-150 cm at the drains. Pond volum es ranged from about 450 to 480 m 3. These ponds have been used annually for experim ents on w ater quality and pond fertilization for 25 years.
Soils used for construction o f these ponds w ere typic, K andiudults (clayey, kaolinitic, and therm ic). Such soils are acidic, reddish brow n and are o f low cation exchange capacity w ith base saturation less than 35%, and in their native state, these soils have low concentrations o f phosphorus and organic m atter (M cN utt 1981).
Ponds are supplied by w ater from a reservoir filled by runoff from w oodland (B oyd 1990). T his w ater has total alkalinity and total hardness values below 20 m g/L as CaCC>3 as well as soluble reactive phosphorus
concentrations less than 0.005 m g/L. These ponds are treated m ost years w ith 500 to 1,000 kg/ha o f agricultural lim estone to m aintain adequate pH, total alkalinity, and total hardness.
Management
The ponds w ere stocked at 15,000 channel catfish fm gerlings (10.7 g/ha) in A pril 1996. A 32% crude protein, pelleted feed w as fed 7 days per w eek at 3% o f bo d y w eight/day. Feeding rates w ere increased w eekly according to an assum ed feed conversion (w eight o f feed applied/w eight gained by the fish) o f 1.6. Ponds w ere inspected daily, and any dead fish w ere rem oved. D aily feeding rate did not exceed 75 kg/ha. W hen this rate w as reached, it w as continued until fish harvest. A ll ponds had a 0.33-kW , vertical pum p aerator (A ir-O -L ator C orporation, K ansas City, M issouri1) connected to a timer. A erators w ere operated from dusk to daw n from the end o f June until harvest in October. W ater levels in ponds w ere m ain tained 1 0 - 1 2 cm below the tops o f standing drain pipes to prevent over
flow after rains. W ater w as added from a pipeline w hen necessary to replace evaporation and seepage losses. Ponds w ere drained, and fish were harvested and w eighed on 15 O ctober 1996.
BOC Treatment
A B O C product, m arketed under the trade nam e A C +, w as obtained from N eozym es International, Inc., A liso Viejo, C alifornia. The experi m ent w as designed so th at the treatm ent applied to any given pond w as not revealed until after the ponds w ere harvested. The researchers assigned each pond a letter (A -I) by ballot. The m anufacturer bottled 960-m L aliquots o f A C + to provide 2 m g/L in ponds (based on 480 m3 volum e)
and equal volum es o f a placebo consisting o f herbal tea that looked like A C+. B ottles w ere identified by the application date and letters A to I to correspond to letters assigned to ponds and shipped to A uburn University.
This provided three treatm ents o f three replications each, as follows: high A C + treatm en t-2 m g/L A C+ w eekly ( 8 m g/L per m onth); low A C + treat
m e n t-2 m g/L A C+ one w eek follow ed by applications o f placebo for three w eeks (2 m g/L per m onth); co n tro l-p laceb o at w eekly intervals. The m anufacturer sent the code for identifying bottle contents to C raig S. Tucker, D elta R esearch and Extension Center, Stoneville, M ississippi,°and he revealed it to the researchers at the end o f the study. These treatm ents w ere applied betw een 13 M ay and 11 O ctober 1996. The placebo and A C + w ere splashed over pond surfaces in the vicinity o f the aerators, and the aerators w ere then operated for 1 hour to m ix the m aterials w ith the pond
waters.
Water, Soil, and Fish Analyses
D issolved oxygen and tem perature w ere m easured one or m ore tim es per day for m anagem ent purposes w ith a polarographic oxygen m eter (Yel low Springs Instrum ent Company, Yellow Springs, Ohio) to verify that fish were not subjected to low dissolved oxygen stress. W ater sam ples were collected between 0630 and 0700 at 2-w eek intervals with a 90-cm w ater colum n sam pler (Boyd and Tucker 1992). Samples were taken from three places in each pond and com bined to give a com posite for analysis. A naly ses w ere conducted for pH (glass electrode), total alkalinity (acidim etry), total hardness (EDTA titration), specific conductance (conductivity m eter)’ chem ical oxygen dem and (potassium dichrom ate-sulfuric acid oxidation)’ biochem ical oxygen dem and (standard 5-day test), soluble reactive p h o s phorus (ascorbic acid m ethod), total am m onia nitrogen (phenate m ethod), chlorophyll a (m em brane filtration, acetone extraction, and spectroscopy)’ and bacterial abundance (standard plate count). Standard protocol was fol lowed for these analyses (Eaton et al. 1995). Nitrate w as determ ined by the N A S reagent method (van Rijn 1993). Before stocking and one w eek before harvest, five soil samples (the upper 5 cm layer) w ere collected from each pond w ith a 5-cm diam eter core sampler. On each sam pling, the samples from a given pond were com bined to m ake a com posite sample. Soil sam ples w ere dried at 60 °C and pulverized in a ham m er mill. They were analyzed for total carbon by an induction furnace carbon analyzer (Leco Corporation, St. Joseph, M ichigan). At harvest time, the follow ing fish production data were collected, num ber and total w eight o f fish, percentage survival, average w eight o f individual fish, and food conversion ratio.
Data Analysis
M eans w ere tested for statistical differences w ith D u n can ’s m ultiple range test. A large degree o f variation is typically encountered in w ater
quality and fish production variables in experim ents conducted in earthen aquaculture ponds (B oyd et al. 1994). B ecause o f this large variation, the am ount o f replication necessary to declare significant differences at P = 0.05 in pond aquaculture experim ents is often prohibitive. Therefore, in this study, m eans w ere different if the probability was P < 0.1.
RESULTS
Total alkalinity and total hardness concentrations in pond w aters were betw een 25 and 30 m g/L in May, and the concentrations increased to 35 to 40 m g/L in October. S pecific conductance o f pond w aters w as w ithin the range 75 to 105 [xmhos/cm, and m orning pH values w ere betw een 7.7 and 8.2. M orning w ater tem perature in ponds increased from 23 °C on 16 M ay to 2 9 ° C on 8 A ugust and declined to 18°C by 14 October. N o influence o f
A C + treatm ent could be detected on pH , specific conductance, w ater tem perature, alkalinity, and hardness.
B ecause ponds w ere aerated nightly, dissolved oxygen concentrations w ere seldom less than 3 m g/L. F rom July to Septem ber, w hen w ater tem perature and feeding rates w ere high, dissolved oxygen concentrations often w ere 1 or 2 m g/L h ig h er in ponds treated w ith A C+ than in control ponds (Figure 1), b u t the differences w ere not statistically significant (P > 0.1). The high A C + and low A C + treatm ents w ere sim ilar in dissolved oxygen concentration. C oncentrations o f soluble reactive phosphorus (SRP) w ere quite sim ilar in all ponds during m ost o f the study, but there were large increases in SR P concentration in the high A C + treatm ent on two dates and in the low A C + treatm ent on one date (Figure 1). O n a few dates, nitrate-nitrogen (N O3— -N) concentration also tended to be higher in
ponds treated w ith A C + than in control ponds, but these apparent differ ences w ere n o t statistically significant. D uring the sum m er, there w as a trend o f greater, b u t statistically insignificant, concentrations o f total am m onia nitrogen (TAN) in A C + ponds than in control ponds (Figure 2). B iochem ical oxygen dem and (BO D ) and chem ical oxygen dem and (COD) concentrations tended to b e low er during the sum m er in ponds treated w ith A C + as com pared to controls (Figure 3). C hlorophyll a concentration (an index o f phytoplankton abundance) tended to be higher during A ugust and Septem ber in control ponds than in ponds o f the A C+ treatm ents. From June through A ugust, there w as a trend o f greater bacterial abundance in control ponds than in A C + ponds (Figure 4). N evertheless, none o f these apparent differences w as statistically significant. Even though trends o f difference in w ater quality betw een control and A C+ ponds w ere detect able at certain tim es, seasonal m eans for treatm ents were sim ilar (Table 1).
FIGURE 1. Means and standard errors of dissolved oxygen and soluble reactive phosphorus (SRP), measured biweekly in earthen ponds in the Fisheries Research Unit, Auburn, Alabama, that were stocked with 15,000 channel catfish/ha between May to October 1996. Ponds were treated with no, low, and high concentrations of bio-organic catalysts amendments (Con trol, Low AC+, and High AC+, respectively).
FIGURE 2. Means and standard errors of total ammonia nitrogen (TAN) and nitrate, measured biweekly in earthen ponds in the Fisheries Research Unit, Auburn, Alabama, that were stocked with 15,000 channel catfish/ha be tween May to October 1996. Ponds were treated with no, low, and high concentrations of bio-organic catalysts amendments (Control, Low AC+, and High AC+, respectively).
3.0 — Low AC+ High AC+ -o— Control O) E 2.0 1.0 0.0
FIGURE 3. Means and standard errors of chemical and biochemical oxygen demand (COD and BOD, respectively) measured biweekly in earthen ponds in the Fisheries Research Unit, Auburn, Alabama, that were stocked with 15,000 channel catfish/ha between May to October 1996. Ponds were treated with no, low, and high concentrations of bio-organic catalysts amendments (Control, Low AC+, and High AC+, respectively).
FIGURE 4. Means and standard errors of chlorphyll a and bacteria count, measured biweekly in earthen ponds in the Fisheries Research Unit, Au burn, Alabama, that were stocked with 15,000 channel catfish/ha between May to October 1996. Ponds were treated with no, low, and high concentra tions of bio-organic catalysts amendments (Control, Low AC+, and High ACT+, respectively).
TABLE 1. Seasonal means ± S E of water quality data collected from channel catfish ponds treated with extracellular enzymes at 4-week intervals (low AC+), weekly intervals (high AC+), and controls. There were three replica tions of each treatment. Means in a row with different letters were significant ly different ( P < .01).
Variables
Treatment
Low AC + High AC + Control
Dissolved oxygen (mg/L) 5.25 ± 0.44a 5.59 ± 0 .41 a 5.26 ± 0.59a Soluble reactive phosphorus (mg/L) 0.039 ± 0.037a 0.038 ± 0.022a 0.020 ± 0.009b Total ammonia nitrogen (mg/L) 0.450 ± 0.191a 0.350 ± 0.185a 0.280 ± 0.187a Nitrate nitrogen (mg/L) 0 .1 8 0 ± 0 .1 5 a 0.130 ± 0.093a 0.090 ± 0.046a Chemical oxygen demand (mg/L) 2 3 .5 ± 5 .1 a 19.0 ± 4.8a 25.1 ± 5.4a Biochemical oxygen demand (mg/L) 7.8 ± 1.5a 7.8 ± 1.5a 8.1 ± 1.5a Chlorophyll a (ng/L) 64.5 ± 23a 54.5 ± 16.7a 62.2 ± 19.7a Bacterial abundance (cells/mL x to 3) 10.5 ± 3.2a 11.7 ± 5.3a 1 3 .0 ± 4 .0 a
T he on ly significant difference w as for SRP concentration, and the differ ence resulted from high concentrations o f SRP in A C + ponds on a few dates.
P ond soils on the FR U do n o t naturally contain free calcium carbonate (M cN utt 1981), and only sm all am ounts o f residual calcium carbonate could have resulted from agricultural lim estone applications. Therefore, the total carbon concentrations in pond soil w ere considered to result p rim arily from organic carbon. In M ay, averages and standard deviations fo r percentages o f soil carbon w ere as follow s: control, 0.81 ± 0 .1 5 ; low A C + , 1.00 ± 0 .3 0 ; high A C +, 0.70 ± 0 .1 8 . Soil carbon increased in all ponds during the study, and percentages found in O ctober were: control, 1.26 ± 0 .4 7 ; low A C+, 1.40 ± 0 .9 1 ; high A C +, 1.21 ± 0 .5 9 . These data suggest that A C + treatm ent did not influence the rate o f increase o f soil organic carbon in pond bottom s.
F ish production data are sum m arized in Table 2. A portion o f the fish in all ponds w ere infected by proliferative gill disease and enteric septicem ia o f catfish, E dw ardsiela ictaluri, and survival ranged from 56.1% in con trols to 75.7% in the high A C + ponds. N evertheless, sim ilar am ounts o f feed w ere applied in all ponds, and at harvest fish were sim ilar in size in all ponds. F eed conversion ratio and net fish production did not differ am ong treatm ents (P > 0.1).
TABLE 2. Mean production d a ta ± S E for ponds treated with extracellular enzymes at 4-week intervals (low AC+), weekly intervals (high AC+), and controls. There were three replications of each treatment. Ponds were stocked at 15,000/ha.
Variables
Treatment
Low AC + High AC + Control
Feed input (kg/ha) 6,128a 6,298a 6,008a
Survival (%) 6 8 .2 ± 8 .6 a b 75.7 ± 7.42a 56.1 ± 6.3b Harvest weight (g) 3 4 2 ± 3 2 a 328 ± 36.8a 400 ± 13.8a Net yield (kg/ha) 3,502 ± 300.8a 3,728 ± 502.8a 3,302 ± 279.2a Feed conversion ratio 1.75 ± 0.12a 1.77 ± 0.0a 1.82 ± 0.12a
DISCUSSION
E ven though pon d s w ere aerated during the night, there w as a trend o f h igher dissolved oxygen concentrations in the ponds treated w ith A C + as com pared to control ponds (Figure 1). This trend in dissolved oxygen concentration w as m ost obvious in A ugust and Septem ber; the trend re sulted because o f the tendency for higher concentrations o f CO D and B O D , and m ore phytoplankton in control ponds than in A C+ ponds caused a greater nighttim e dem and for oxygen in control ponds during A ugust and Septem ber. N evertheless, w hen phytoplankton abundance and con centrations o f dissolved oxygen, C O D , and BO D w ere averaged over all dates, they did not differ (P > 0.1) am ong treatm ents (Table 1).
O ne explanation for the low er oxygen dem and at tim es in A C+ ponds is that additions o f B O C s accelerated the decom position o f organic m atter to cause low er B O D and CO D concentrations. This explanation is consistent w ith the observation that SRP, TAN, and N0 3~ -N concentrations som e
tim es tended to be greater in A C + ponds (Figures 1 and 2), because greater decom position w ould have caused m ore nutrient recycling. How ever, greater nutrient recycling should have stim ulated phytoplankton produc tivity in the A C + ponds, but this effect w as not observed. A lso, sim ilar rates o f organic carbon accum ulation in pond soils o f the three treatm ents is not consistent w ith enhanced decom position o f organic m atter in A C + ponds.
A m ore feasible explanation for the apparent trend in higher concentra tions o f chlorophyll a, B O D and CO D at tim es in the control ponds is that A C + reduced the phytoplankton grow th rate. Boyd (1973) and B oyd et al.
(1978) dem onstrated that BO D and COD concentrations in pond waters had a strong positive correlation w ith chlorophyll a concentrations (phyto plankton abundance). A n increase in chlorophyll a concentration should cause an increase in B O D and C O D concentrations. N utrient inputs to all treatm ents in feed w ere sim ilar, and the tendency o f greater nutrient con centrations in A C + ponds relative to controls could have resulted from less uptake o f nutrients b y sm aller phytoplankton biom ass in A C + ponds instead o f greater rates o f nutrient recycling in A C + ponds. B oyd and M usig (1980) show ed th at the rate o f decline in spikes o f SRP m ade to pond w aters increased w ith increasing phytoplankton abundance. Tucker et al. (1984) dem onstrated that TA N concentrations in fish pond w aters decreased as phytoplankton abundance increased. Thus, the apparent trend in low er nutrient concentrations and in higher BOD and CO D values in control ponds is consistent w ith the explanation that A C + reduced the phytoplankton abundance. F urther support o f the effects o f A C + on phyto plankton grow th rates com es from the claim by the m anufacturer that AC+ can be used to reduce the abundance o f nuisance algal grow th in water. B ecause A C + is a proprietary product, its com position w as n o t divulged by N eozym es International. W ithout this inform ation, no speculation can occur on the m echanism by w hich A C + m ight have influenced phyto plankton growth.
D ifferences in fish survival w ere observed (Table 2). This result could be related to the actions o f the A C + ; however, because o f the high varia tion am ong the ponds, the lim ited num ber o f replicates, and the disease problem s that w ere observed, farth er investigation o f the effects o f A C + is needed.
T he apparent im provem ent in dissolved oxygen concentration in AC+ ponds did not cause greater fish production. This is not surprising because the stocking rate w as conservative and the feeding rate did not exceed 75 kg/ha per day. W hen m echanical aeration is used, dangerously low dis solved oxygen concentrations, excessive TAN concentrations, and other w ater quality im pairm ent seldom occur unless feeding rates exceed 100 to 120 kg/ha per day (B oyd 1990; T ucker and B oyd 1995). The likelihood o f w ater quality im provem ent and enhanced fish production through the use o f B O C supplem entation w ould be greater in ponds w here stocking and feeding rates are high enough to cause m ore severe w ater quality deterio ration than w as observed in the p resent study.
This study show s that B O C ’s supplem entation did not seem to im prove w ater quality even in aquaculture ponds stocked and fed at conservative rates. Further studies are needed to show if greater benefit can be achieved in ponds w here stocking and feeding rates are higher and w ater quality
conditions are m ore critical. Inform ation on the m echanism s o f action o f extracellular enzym e additions in ponds w ould also be useful in assessing the potential benefits o f these products and defining the conditions under w hich they can be effective.
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