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TOXIC EFFECTS OF ZINC FROM TROUT FARM SEDIMENTS ON ATP,

PROTEIN, AND HEMOGLOBIN CONCENTRATIONS OF LIMNODRILUS
HOFFMEISTERI
Laura Martinez-Tabche ; Irma Gutierrez Cabrera ; Leobardo Gomez Olivan ; Marcela Galar Martinez
;Celia German Faz

To cite this Article Martinez-Tabche, Laura , Cabrera, Irma Gutierrez , Olivan, Leobardo Gomez , Martinez, Marcela Galar
andFaz, Celia German(2000) 'TOXIC EFFECTS OF ZINC FROM TROUT FARM SEDIMENTS ON ATP, PROTEIN, AND
HEMOGLOBIN CONCENTRATIONS OF LIMNODRILUS HOFFMEISTERI', Journal of Toxicology and Environmental
Health, Part A, 59: 7, 575 — 583
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 Journal of T oxicology and Environm ental Health, Part A, 59:575±583, 2000
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TOXIC EFFECTS OF ZINC FROM TRO UT FARM SEDIMENTS

ON ATP, PROTEIN, AN D HEMOG LOBIN CONCENTRATIONS
OF LIM N O D RILU S H O FFM EISTERI

Laura Martinez -Tabche, Irm a Gutié rrez Cabrera,

Leobardo Gómez Olivá n, M arcela Galar Martine z, Celia Germán Faz
Departam ento de Toxicología, Sección de Graduados, Escuela
Nacional de Ciencias Biológicas, Institu to Politécnico Nacional,
Delegación M . Hidalgo, México
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Zinc ( Zn) is a nutritionally essential m etal, and deficiency results in severe health conse-
quence s to aq uatic organism s. In this study toxicity dat a for Limnodrilus hoffm eisteri
prod uced b y Zn in system s using three natural sed iment s ( trout farms: El O yam el, El
Truchón, and El Potrero) are presented. H em oglob in, ad enosine triphosphate ( ATP) , and
protein concent rations were m easured in L. hoffm eisteri exposed to sp iked sediments, as
ind icators of exposure. Physicoc hem ical characteristics of water and sedim ents were also
consid ered. Zn co ncent rations w ere m easured in w ater and sed iment. El O ya m el, El
Truchón, and El Potrero pond sedim ent s did not have sim ilar p hysicoch em ical character-
istics. Zn concen trations of water obt ained from the rustic p onds were near 0.4575 m g/
L; how ever, this m etal w as always found to be higher in the sediments ( 0.0271– 0.9754
m g/ kg) . The b ioassay with worm s d em onstrated that p ond sed iments from El O yam el, El
Potrero, and El Truchón prod uced toxicity since ATP and p rotein concen trations were
low com p ared to controls ( organism s without m etal) . All spiked sedim ents had a signifi-
cant reduction effect on ATP, protein, and hem oglob in concentrations. This investigation
clearly show s that sedim ent s of El Truchón, El O yam el, and El Potrero possess toxicity
potential. These results suggest the usefulness of these bioassay s to evaluate the toxicity
of sedim ents polluted with heavy m etals.

The trout ( Onchorhynchus mykiss ) was introd uced alm ost a century
ago to central M exico. Cu rre ntly this fish is cultured in m any M exican
states, w ith the Estado de M éxico, being the site of the most production
(90% ). H ow ever, according to the Fisheries D epartm ent, production dim in-
ished in 1 992 by 14 % (A nuario Estad ístico de Pesca, 199 2). The m ain
problem reported by aquaculturists w as related to the viability of young
fish. Culturing of the trout was carried out in concrete ponds or earthen
raceway ponds. These holes w ere m ade in the ground w ithout any cover-
ing; hence w ater quality is affected by accum ulation of detritus and sedi-
ments. Previous studies done in our laboratory demonstrated that N i as
w ell as Zn w ere the p rincipal pollutants found in the sed im ents o f the
reservoirs of this farm (G utiérrez et al., 1997).

Received 27 M ay 1999; sent for rev ision 8 July 1999; accepted 19 O ctober 1999.
Addre ss corresp ondenc e to Dr. Laura M artínez-Tabche, Departam ento de Toxicología, Sección
de Graduad os, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nac ional, Delegación
M . Hidalgo, CP 11340 D.F., M éxico. E-m ail: ltabche@ bios.encb.ipn.m x

575
 576 L. M ARTIN EZ-TA BCHE ET AL.

Oligochaetes play a m ajor role in the processing of organic material,

are often associated w ith aquatic systems rich in organic m atter, and are a
food source for benthic feeding fish. These organism s reside in sediments
and consum e food containing this substrate (Khangarot, 1991); therefore,
these w orm s h ave d irect co ntact w ith xenob iotics, and can b e used in
toxicity studies, Ross and M unaw ar (1996) suggested that Tubifex tubifex
is an organism of choice to evaluate sediments toxicity.
Zn found in natural form in the environment is present in quantities
that are relatively sm all. The norm al content of this m etal in soils is 0.5
mg/kg, in sea w ater 0.005 m g/kg, and in vegetables from 1 to 50 m g/kg.
This m etal is usually associated w ith Cd and Pb. Zn is released into the
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atm osphere in oxide form . Zn is a nutritionally essential m etal, and defi-

ciency results in severe health consequences in organism s (H ostýnek et
al., 1993). H ow ever, there is evidence that this metal produces adverse

trations inhibited d -aminolevulinase m ore than Pb. This enzyme partici-

effects on hem atology. Bull (1980) dem onstrated that Zn in high concen-

pates in the synthesis of hem e. Further, Zn m ay affect other proteins. The

stress protein response appears to serve as a valid biomonitoring m ethod
for chronic and sublethal exposures, prior to biological responses becom -
ing overtly evident in a population, but may not be a realistic indicator
when the stressor intensity is at or near lethality (Nascimento et al., 1998).
Rosenthal and Alderdice (1976) dem onstrated that in fish exposed to high
concentrations of this metal, decreased levels of ATP w ere found. Consid-
ering the structure of the ru stic reservoir (which is never w ashed), and
that food given to fish contains Zn, w hich is also present in detritus and
sediments, the reservoirs contain h igh concentrations of this m etal. The
objective of this study was to determ ine whether Zn produced toxicity as
measured in term s of protein, adenosine triphosphate (ATP), and hem o -
globin concentrations in Limnodrilus hoffm eisteri and to determ ine w hich
of these sediments w as m ore toxic.

MATERIAL AND M ETHODS

This study was perform ed at three trout farm s: El Oyamel, El Potrero, and
El Truchón, located 32.5 km east of Toluca, Estado de México, in Ocoyo-
acac M unicipality near Guadalupe Victoria D am. These trout farm s receive
w ater fro m Paja rito s an d A gu a A zu l w ells a t a rate o f 10 L/s (A n ua rio
Estadístico de Pesca, 1992). Sampling was carried out between February and
March 1997 (beginning and end of February and end of March). Pond aver-
age depth was 1 m, w ith an average surface area of 23.8 m 2. Water samples
were taken from one pond at each trout farm to evaluate physicochemical
characteristics. Triplicate samples were collected in sterile polyethylene bot-
tles from the surface water layer at each site. Water temperature, pH, and
dissolved oxygen concentration at the surface w ere determ ined at the site
using an oxymeter (Simpson Electric CO.YST model 51B). The concentra-
tion of nitrites and am monium w ere determ ined by colorim etric method
 TO XIC EFFEC TS O F ZIN C O N LIM N O DRILU S 577

using a spectrophotometer (Hach D REL model 2000), while hardness (cal-

cium carbonate) was measured by ethylenediamine tetraacetic acid (ED TA)
titration. The Zn concentrations w ere determ ined by atom ic absorption.
Three sediment samples of each pond were taken w ith a 5.2 cm × 6 cm
diameter conical dredge of stainless steel, stored in acid-washed polyethyl-
ene bottles in the dark, cooled w ith ice during transport to the laboratory,
dried at constant w eight at 80°C, and then ground w ith a porcelain mortar.
Sedim ents w ere characterized by determ ination of granular com position,
total nitrogen, organic matter content, pH , and Zn concentrations.
Limnodrilus hoffmeisteri was collected from Texcoco Lake, M éxico (from
natural sources), and held under laboratory conditions for 7 d prior to experi-
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ments. In the laboratory, worm s w ere placed in sand (particle size = 0.84
mm), fed with molasses (5% ), diluted w ith culture water (pH 7.6; alkalinity
48.4 mg HCO 3/L; Ca 2+ 80 mg/L), and maintained at 6.4–6.6 mg/L dissolved
oxygen and 21 ± 0.5°C. Surface sediments w ere obtained at 1 m depth from
one pond each at El Oyamel, El Potrero, and El Truchón; the control was
sand without Zn spiking. Zn [Zn(NO 3) 2] was spiked in each sediment sample
at three concentrations (0, 1, or 2 mg/kg), The spiked sediment was mixed
for 20 min and equilibrated for approximately 2 h. These concentrations
were based on reported threshold concentrations for fish (0.2–2 mg/L at 96 h)
(M etelev et al., 1983) and worm s (CL50/10 = 2.56 mg/kg at 96 h) (Khan-
garot, 1991). Then 2.5 g of worm s (wet weight) was added to each poly-
styrene cup containing 1 g of spiked sediment (El Oyamel, El Potrero, and El
Truchón) reconstituted with 100 ml of culture water. After 72 h of exposure,
1.0 g of L. hoffmeisteri (wet w eight) were hom ogenized. The homogenate
was centrifuged at 7500 g × 5 min at 4°C, the pellet was discarded, and pro-
tein, ATP, and H b concentrations w ere determined in the supernatant.
Protein, ATP, and H b w ere measured using methods of Bradford (1976),
Adam s (1963), and W introbe (1958), respectively. Zn m easurem ents w ere
m ade according to the follow ing protocol: For sediment 1 g sam ple of
sediment was w eighed. For water sam ples 1 m l of water was taken. Each
of these was m ixed w ith 10 m l of concentrated nitric acid. These sam ples
w ere heated in an au toc lave fo r 0 .5 h at 12 1°C ; Zn w as an alyzed by
atom ic absorption spectroscopy w ith a Varian m odel AA-1475. Zn con-
centration in the sediments and water was calculated on mg/kg dry w eight
basis and mg/L, respectively.
Statistics
Data w ere analyzed by analysis of variance (ANOVA). The significance
of differences betw een groups w as tested using the D uncan m eans test.
The criterion for significance w as p < .05.

RESULTS
Water quality and chemical conditions m easured during the study are
presented in Table 1. Levels of dissolved oxygen and hardness in the water
of ponds in M exico w ere low er than those in other lakes w here trout pro-
 578 L. M ARTIN EZ-TA BCHE ET AL.

TA BLE 1. Physicochem ical Characteristics of Water From Three Trout Farm s of M éxico State

Site
__________________________________________
Parameter El O yam el El Potrero El Truchón

Tem perature (°C) 14 13 14

Hardness (CaCO 3 ) (mg/L) 96.0 ± 2.33 99.5 ± 3.6 101.0 ± 7.5
Am monia (mg/L) 0.66 ± 0.001 a 0.84 ± 0.006 a 0.105 ± 0.005 a
Oxygen (mg/L) 9.5 ± 0.3 10 ± 0.09 10 ± 0.085
Nitrites (mg/L) ND ND ND
pH 7.3 ± 0.5 8.1 ± 0.08 7.1 ± 0.03
Zn (mg/L) ND 0.457 ± 0.009 0.0472 ± 0.01
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N ote. Each value represents the average of three replicates ± SD. ND, not detected.
a
Significant at p < .05.

duction was found successful (Sly & Evans, 1996). M ost of the water qual-
ity condition s m easured during this study appeared to be adequ ate for
supporting trout culture. H ow ever, other potentially stressing conditions
su ch as am m on ia w ere fou nd in th e w ater sam ples o f M exican p on ds
(Table 1). In these trout farm w aters, amm onia concentrations w ere above
the recom mended levels (0.08 m g/L) (Ackefors et al., 1994).
G eneral physicochem ical characterization of experim ental sediments
is given in Table 2. El O yam el, El Potrero, and El Truchón pond sedim ents
did not have sim ilar physicochem ical characteristics. There w ere m ajor
differences betw een El O yam el and El Potrero versus El Truch ón in all
para m eters e xcep t p H an d p erc en t clay. Th e co nce ntratio n o f Zn w as
markedly different at all three sites, being low est at El O yam el, 10-fold
higher at El Truchón, and 40-fold greater at El Potrero.
D ata in Figure 1 show that ATP levels in L. hoffm eisteri w ere signifi-
cantly low er in natural pond sedim ents com pared to control (Zn-deficient
sediment). Spiking the sedim ents w ith Zn at 1 or 2 m g/kg resulted in fur-
ther reduction in w orm ATP levels.

TA BLE 2. Physicochem ical Characteristics of Sedim ents From Three Trout Farm s of M éxico State

Site
____________________________________________
Parameter El O yam el El Potrero El Truchón

Organic m atter (%) 30.33 ± 3.5 32.6 ± 0.38 10.18 ± 0.351

Clay (%) 9.71 ± 2.8 10.98 ± 1.65 12.42 ± 0.59
Silt (%) 30.8 ± 2.5 36.90 ± 1.11 12.16 ± 0.613
Sand (%) 54.12 ± 5.3 50.53 ± 0.95 75.40 ± 0.853
Ph 6.12 ± 0.08 6.53 ± 0.14 6.38 ± 0.23
Total nitrogen (%) 5.31 ± 0.21 6.35 ± 0.017 1.47 ± 0.16
Zn (mg/kg) 0.027 ± 0.02 0.9754 ± 0.04 0.3626 ± 0.03

N ote. Each value represents the average of three replicates ± SD.

 TO XIC EFFEC TS O F ZIN C O N LIM N O DRILU S 579
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FIGU RE 1. Effect of Zn on ATP concen trations of L. hoffm eisteri in spiked sedim ents of trout farms
expose d for 72 h. C, sand without m etal; PS, natura l sedim ent; PS + 1, natural spiked sedim ent w ith
1 m g/kg of Zn; PS + 2, natural spiked sedim ent w ith 2 m g/kg of Zn; (| ) El O yam el sedim ent; ( m ) El
Potre ro sedim ent; ( M ) El Truchó n sedim ent. Each value represents the ave rage of three replicates ±
SD. Asterisk indicates significantly different from control (p < .05). Plus sign indicates significantly
different from natural corresp onding sedim ent (p < .05).

The sedim ent at all three sites did not m arkedly affect w orm protein
levels (Figure 2). Spiking w ith Zn also did not affect protein concentra-
tions of worm s at El Potrero. H ow ever, a significant fall in w orm protein
levels w as seen at El Oyam el and El Truchón.
D ata in Figure 3 show a significant fall in H b content in w orm s at El
Potrero. Spiking of sedim ent w ith Zn from all three sites significantly de-
creased worm H b content. Worm s at El O yam el appeared least affected.

DISCUSSION
Th e m ain n itro gen o u s e xcreto ry p rod u ct o f m o st fish is am m o n ia,
which is discharged into the water through the gills. In addition, dissolved
urea and particulate waste (food) in the water are converted chem ically
and/or microbiologically to inorganic com pounds such as amm onia and
nitrite (Ackefors et al., 19 94 ). In M exican trou t farm w aters, am m o nia
concentration was above the recomm ended levels, which m ay be suffi-
cient to cause stress and could be the basis for trout production decline.
The threshold effect lim it (TEL) level for Zn in water is 0.1–2 m g/L
(M etelev et al., 1983) and in sedim ents 123 m g/kg (Sm ith et al., 1996).
 580 L. M ARTIN EZ-TA BCHE ET AL.
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FIGU RE 2. Effects of Zn on protein concen tration of L. hoffm eisteri in spik ed sedim ents of trout farm s
expose d for 72 h. C, sand without m etal; PS, natura l sedim ent; PS + 1, natural spiked sedim ent w ith
1.0 m g/kg of Zn; PS + 2, natural spiked sedim ent w ith 2.0 m g/kg of Zn; ( | ) El O yam el sedim ent; ( m )
El Potre ro sedim ent; (M ) El Truchó n sedim ent. Ea ch value represents the average of three replicates ±
SD. Asterisk indicates significantly different from control (p < .05). Plus sign indicates significantly
different from natural corresp onding sedim ent (p < .05).

The Zn concentrations found in this study are low er than the TEL in both
media and thus w ould not be expected to be toxic. H ow ever, sedim ent
from El O yamel containing 0.027 m g/kg Zn significantly decreased ATP
concentration in w orm s. The highest concentration of Zn occurred near
the indu strialized area o f Toluca. D ata d o not ind icate how this m etal
moved from original sources to deposition areas, but it was suggested that
Zn w as absorbed by sediment particles and carried dow nstream by river
curre nts. Since Zn is an essential elem ent, biotic processes often enhance
the accum ulation of this metal in sediments, especially during suspension
feeding (D íaz & Rosenberg, 1996).
Adenosine triphosphate content was severely reduced in worm s ex-
posed to El Potrero spiked sedim ent (this trout farm has the highest con -
centration o f Zn). The d ecrease m ay b e attribu table to d irect effect of
metal on mitochondrial respiration. Inhibition of som e enzym e systems
probably occurred (Tort et al., 1982). Zn significantly decreased H b level
in all spiked sedim ents and w as highest in El Potrero sedim ents. It is of
in te re st th a t in o u r la b o ra to ry (n o t re p o rte d ) w ith cla d o c e ra n M oina
macrocopa expo sed to different Zn con centratio ns, H b co ncen tration s
w ere red uced significan tly co m pared to con tro l. Anem ia is a co m m on
sym ptom of sublethal Zn intoxication in mam mals, since this metal limits
 TO XIC EFFEC TS O F ZIN C O N LIM N O DRILU S 581

the absorption of copper and iron; therefore it m ay alter hem oglobin syn-
thesis in worm s (Leland & Kuw abara, 1985).
The protein content w as m odified in all the treated sedim ents as com -
pared w ith natural sedim ents. M ehrle and M ayer (1984) reported altered
protein concentration in fish exposed to heavy metals. This biochemical
change indicated hem oconcentration and impaired w ater balance (Lew is
& Lew is, 1971). The w orm s exposed to El Potrero spiked sediments did
not disp lay change in p rotein con centration , su ggesting that this effect
may be due to induction of m etalloprotein synthesis. W inge et al. (1975)
reported a low -m olecular-w eight Zn-binding protein cytosol follow ing Zn
injection. It is not clear w hat role all these proteins may play, but in this
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specific case it could be a defense mechanism against Zn toxicity, since

these organism s w ere exposed to very high concentrations of Zn, but the
precise mechanism needs further investigation. The sedim ents tested m ay
also have other m etals such as N i (M artínez-Tabche et al., 19 99), that
may increase this response. Further earthen raceway ponds contain amm o-
nia, which can contribute to increase or m odify toxicity. Amm onia is cer-
tainly toxic to catfish brain, w here inhibition of ATP production in nerve
cells occurs (Tom asso et al., 1980).

FIGU RE 3. Effects of Zn on hem oglobin concen tration of L. hoffm eisteri in spiked sedim ents of trout
farms expose d for 72 h. C, sand w ithout m etal; PS,natural sedim ent; PS + 1, natural spiked sedim ent
with 1.0 m g/kg of Zn; PS + 2, natural spiked sedim ent with 2.0 m g/kg of Zn; (| ) El O yam el sedim ent;
(m ) El Potrero sedim ent; (M ) El Truchó n sedim e nt. Each value represents the average of three repli-
cates ± SD. Asterisk indic ates significantly different from control (p < .05). Plus sign indicates signifi-
cantly different from natural corresp onding sedim ent (p < .05).
 582 L. M ARTIN EZ-TA BCHE ET AL.

In conclusion, the three sedim ents of the reservoirs in this study w ith-
out Zn spiked w ere toxic to the worm and therefore can be toxic for trout,
since the pollutants stored in the sediments are in equilibrium w ith the
water of the reservoir. Power and Chapman (1992) recognized that aquatic
sedim ents m ay p lay an im portant role in storing con tam inan ts fro m, o r
releasing them into, the w ater column.
Our study documents that the biochem ical parameter most sensitive to
Zn effects is ATP concentrations, which can be detected by L. hoffmeisteri
assays for potential toxicity. These results support the use of this bioassay in
the evaluation of environmental contamination, since it is a sensitive and
rapid assay for routine analysis.
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