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Philippine Journal of Science

138 (1): 91-104, June 2009

ISSN 0031 - 7683

Assessing the Impact of Fish Cage Culture on

Taal Lake (Philippines) Water and Sediment
Quality Using the Zebrafish Embryo Assay

Arnold V. Hallare1,2, Patricia Ann Factor1, Erica Katrina Santos1, and Henner Hollert2

Department of Biology, College of Arts and Sciences,
University of the Philippines, Manila, Padre Faura, Manila, Philippines
Department of Ecosystem Analysis, Institute for Environmental Research,
RWTH Aachen University Worringerweg 1 D-52074 Aachen, Germany

The present study investigated the impact of fish cage aquaculture on the quality of lake water and
sediment using a contact assay with zebrafish (Danio rerio) embryos. Fertilized zebrafish embryos
were exposed to surface waters and whole sediments collected from three sites of varying levels of
stress from Taal Lake, Philippines. Gonzales was the reference site, while Baaga and Balakilong
were sites of heavy aquaculture. No significant developmental differences were observed in water
samples collected from all sites. Very high survival rates, high hatching success rates, and few
abnormalities were observed in all water samples. All values did not differ significantly from the
negative control. Similarly, zebrafish embryos exposed to sediments from the reference site and
from sediment control also developed normally and showed no significant defects. However, a
significant reduction in both survival and hatching success rates, and a considerable increase in rates
of abnormalities, were recorded from sediments obtained from the aquaculture sites. The observed
embryotoxic responses were discussed in relation to the presence of contaminants such as ammonia
and copper which have settled in the sediments from unconsumed feeds. Results of the study clearly
demonstrate that fish cage culture has a harmful impact on lake quality (particularly the sediment
phase). We also recommend the use of an effect-directed analysis as a next-level approach to identify
specifically other individual toxicants responsible for the teratogenic effects.

Key Words: ammonia, embryotoxicity, fish cage, heavy metals, sediment quality, Taal Lake,

INTRODUCTION out that the prevailing winds and thermal stratification

of the lakes deep water brought heavy loads of organic
Philippine lakes are constantly being threatened with sediment to the surface. This resulted in oxygen depletion,
various stressors such as unregulated open fishing, heavy and raised levels of ammonia, hydrogen sulfide, and nitrite
metal pollution, agricultural and domestic pollution and levels in the lake, contributing to the fish kill. The large
more recently, by fish cage aquaculture (Dela Vega 2001; organic load in the lake could be traced to excess feed
Yambot 2000; Vista et al. 2006). The reported fish kills and fish wastes from tilapia cages that have proliferated
in Taal Lake suggest a deteriorating lake quality (Yambot in the lakes of Agoncillo, Leviste, and Laurel in Batangas
2000). From May 1998 to June 2001, a total of 38 fish City. The unconsumed food from fish cage aquaculture
kills in various areas close to aquaculture cages were have been shown to settle at the bottom of the lake. Its
reported (Rosana and Salisi 2002). Researchers pointed degradation and interaction with several other pollutants
*Corresponding author: avhallare@post.upm.edu.ph from domestic, industrial, and agricultural wastes are
Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake

endangering the aquaculture industry and the Taal Lake The objectives of the present study were: (1) to assess the
ecosystem as a whole (Dela Vega 2001). Until now, there impact of fish cage culture on water and sediment quality
have been no studies addressing the potential link between of Taal Lake, Philippines using biomarker responses
heavy aquaculture activities and the worsening health state in zebrafish embryos, (2) to compare the embryotoxic
of the Taal Lake ecosystem. potential of waters and sediments, (3) to determine if
there is a gradient of toxicity among the different sites in
It has been widely accepted that, for a complete appraisal Taal Lake. While it is acknowledged that the responses
of aquatic ecosystem health, monitoring and assessment of adult fish to probable contaminants may be quite
should not focus on surface water alone but must include different, determination of embryotoxic responses offers
sediment toxicity analysis (Burton 1991; Brunstrm et much broader ecotoxicological relevance. In addition,
al. 1992; Ahlf et al. 2002; Hollert et al. 2003). Sediments many adult pelagic and epibenthic organisms depend on
not only act as sinks for pollutants but also as secondary sediments as food source and breeding substrates such that
sources for many persistent chemicals (Braunbeck the effects on reproductive behavior, embryo development,
et al. 2005). Although there is a monthly monitoring hatchability, and growth are critical endpoints to consider
conducted by the Bureau of Fisheries and Aquatic (Burton 1991).
Resources (BFAR, Philippines), this is usually limited
to testing surface water and the determination of its
physico-chemical characteristics. Chemical monitoring
is valuable, but it can only offer little information on the
actual and impending biological activity/availability of
contaminants. To ensure a more significant evaluation of
lake water and sediment quality, their effects on resident The Study Site
biota must be studied and assessed. The determination For this study, three barangays along Taal Lake were
of biomarker responses such as mortality, abnormalities, selected as sampling sites based on probable pollution
and hatching success in organisms can serve as more gradient: Gonzales (Gon) which is located in Tanauan
precise indicators of the health status of the aquatic City, Baaga (Ba) in the municipality of Agoncillo,
environment than the mere presence or absence of and Balakilong (Bal) in the municipality of Laurel
chemicals (Hahn 2002; Hallare et al. 2005a). Biomarkers (Figure 1). These sites were likewise recommended by
can serve as early warning signals before populations the Bureau of Fisheries and Aquatic Resources (BFAR).
and communities are severely damaged (Triebskorn et Gonzales is a site of open water fishing, while Baaga
al. 1997). and Balakilong are sites of heavy aquaculture. In 2006,
BFAR reported the presence of 1,024 fish cages in Baaga
The developing fish embryo is generally considered and around 2,305 fish cages in Balakilong area. Total fish
to be the most sensitive stage in the life cycle of fish production in the same year showed a value of 48,896.3
(Ozoh 1980a; Kristensen 1995; Luckenbach et al. MT in high aquaculture sites compared with only 7,391
2003). Previous studies have documented the greater MT in open water fish catch (BFAR 2008). Several sets
sensitivity of fish embryos and larvae to chemicals of physicochemical data were obtained at each site in
compared with adults (Kristensen 1995; Nagel 2002; October 2006. In addition, composite surface water
Hollert et al. 2003). The increasing interest in the samples from each site were collected in polyethylene
so-called fish embryo test in Europe and the United plastic jars and stored on ice until chemical analyses and
States stems from its validation for use in standardized bioassays were undertaken. Composite surface sediment
wastewater assessment in Germany (DIN 38415-6) and samples (0-5 cm) were also collected from each site using
its submission in a modified version to the Organization a sediment grab. Sediment samples were stored in pre-
for Economic Cooperation and Development (OECD) washed and labeled polyethylene jars and stored on ice
guidelines. It is now regarded as a suitable alternative prior to chemical testing and processing. Wet sediment
to the acute (adult) fish test, which elicits severe ethical samples were freeze-dried at -30 C, and were stored in
considerations and provides very limited data (only polyethylene jars at 4 C in the dark prior to bioassays.
LC50) (Nagel 2002; Braunbeck et al 2005). The early
life stage test, on the other hand, is not subject to much Physicochemical analyses of water samples
ethical concerns and also offers various toxicological (temperature, salinity, conductivity, pH, and dissolved
endpoints for determining adverse effects. The gathered oxygen) were obtained in situ using a multiparametric
data could then be used to estimate pollutant toxicity both water analyzer (Horiba, U20XD, Japan). Water and
at the individual and fish population levels (Ensenbach sediment samples were also transported immediately
and Nagel 1997). to the testing laboratory (BFAR Region IV Ambulong,

Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake

Figures 1. Map of Taal Lake showing the location of study sites. Gonzales was the reference site while Balakikong and Baaga were the two fish
aquaculture sites.

Batangas City) for the analysis of ammonia. For tests et al. 2006). Copper from fish feed enters the lake
on heavy metals, water samples were first filtered using environment in uneaten feed or fish feces. Also when
nitrate filter papers (0.45m pore size) and preserved there is more food than is actually consumed by the
by adding 5 mL concentrated nitric acid per one liter fish, it tends to settle at the bottom where it degrades,
of sample to maintain the pH below 4.0. The bottles resulting in increased ammonia which is toxic to fish
were put back in the deep freezer at 4 C until they were embryos (De la Vega 2001).
sent to the Institute of Chemistry Analytical Services
Laboratory of the University of the Philippines-
Maintenance of parental fish
Diliman. The selection of test chemicals, primarily
Adult zebrafish (Danio rerio) were obtained from
copper and ammonia is based on their relevance to
Bioresearch, Inc. and were kept in 15 L glass aquaria under
aquaculture. Copper is usually added to aquaculture
the following conditions: water temperature at 26 0.5
feed to meet dietary requirements of fish. However,
C, hardness at 379 mg/L CaCO3, conductivity at 744 S/
previous research has found that some feeds contain
cm, pH at 7.36 0.2, and dissolved oxygen at 10.5 0.5
excessively high levels of copper (Mendiguchia
mg/mL O2; 95% saturation. They were fed twice daily

Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake

with commercially available feeds (TetraMin flakes) Treatment means that met the parameter assumptions of
and Tubifex worms. The photoperiod was adjusted to 12 h normality and homogeneity were then compared with the
light and 12 h dark. On the evening before spawning, several control using One-Way Analysis of Variance (ANOVA),
rectangular wire mesh boxes were laid on the aquarium floor and this was followed by Dunnetts test. (Microsoft Excel-
to collect the eggs. Spawning was triggered once the light has Analyze It) at p < 0.05 (*) and p < 0.01 (**).
turned on and was completed within 30 minutes.

Exposure conditions
The eggs were collected and rinsed several times with
tap water to remove any debris. Once the four-cell stage
is reached (1-2 h after fertilization), fertilized eggs Water quality parameters
were selected and directly transferred to the exposure The physico-chemical parameters of Taal Lake waters
wells (stabilized to 26 C). Ten eggs in blastula stage are shown in Table 1. The temperature, salinity, and
were transferred to six well-microtiter plates containing conductivity readings for all three sites were typical for
undiluted water samples from the different sites. Artificial a tropical freshwater lake. The pH values were slightly
water (DIN-38415-6; stock solution: 58.8 mg/L CaCl2 x above the set criterion for the Class C status (suitable for
2H2O; 24.6 mg/L MgSO4 x 7H2O; 12.6 mg/L NaHCO3; fishery propagation), of 6.65-8.5. The dissolved oxygen
5.5 mg/L KCl: stock solution diluted to 1:5; ventilated (DO) concentrations were far above the minimum limit
to 100% oxygen saturation prior to the biotest) served for aquatic life which is set at 5 mg/L. For all three
as negative control, while 2.0% EtOH was used as sites ammonia was not detected. The result for heavy
positive control (Hallare et al. 2006). The experiment was metals, particularly for cadmium and lead, was similar
performed three times over a two-month period with three in all three sites with values less than 0.05 mg/L and
replicates per round. 0.10 mg/L, respectively. Copper concentrations were
minimal and variable. Based on the chemical analysis
Freeze-dried whole sediments were mixed with artificial of the lake water, the overall water quality of Taal Lake
sediment (silica powder, grain size W4, Quartzwerke, was maintained at Class C status, making it suitable for
Frehen, Germany) to yield the following concentrations: growth and propagation of fish according to standard
100%, 75%, 50%, and 25%. The total weight of the set by the Department of Environment and Natural
resulting mixture was 3 g. The mixtures were homogenized Resources (DENR 1990).
in a mortar to avoid sediment and silica powder hotspots and
then transferred into six-well microtiter plates. Each well
was filled with 7 mL artificial water, which was previously Sediment quality parameters
ventilated to 100% oxygen saturation. The negative control The physico-chemical parameters of Taal Lake
used was silica (3 g silica + 7 mL artificial water). For sediments are also shown in Table 1. The pH for all
positive control, 7 mL of 2% EtOH was used. The sediment three sites was the same with a value of 6.5. However,
was allowed to settle for 1 h before adding 5 eggs per sample the total ammonia detected varied greatly among sites
per well. The experiment was also performed three times (Balakilong 404.2 ppm; Baaga 239.1; Gonzales
over a two-month period with three replicates per round. 47.12 ppm). Balakilong and Baaga significantly
exceeded the Ministry of Environment and Energy of
Ontarios probable effect level of 100 ppm ammonia
Prolonged zebrafish embryo test (96 h) for aquatic life (MEEO 1993). The unionized ammonia
Progression of embryonic development was monitored was computed according to USEPA (1992) and yielded
at specific times (0, 12, 24 36, 48, 60, 72, 84, and 96 the following values: Balakilong - 0.73 ppm, Baaga -
h) for all exposure groups. The variables used were 0.43 ppm, Gonzales - 0.08 ppm. Again, both Balakilong
egg mortality and hatching success. Malformations and and Baaga exceeded the criteria for unionized NH3
inhibitory tendencies were also described and analyzed. which is set at 0.18 ppm. Cadmium concentration for
These developmental endpoints were studied under the all sampling sites was the same, with a value of less
stereomicroscope (Leica MZ16 A; Meyer Instr Inc.) and than 1.25 mg/kg. Lead concentration, however, varied.
pictures were taken using a digital camera. Baaga contained the highest concentration (11.75 mg/
kg), followed by Balakilong (8.5 mg/kg). Gonzales
Statistical analysis had the lowest concentration (3.5 mg/kg). Copper
Percent mortality, percent abnormality, and hatching success concentrations measured 80.75 mg/kg for Balakilong, 70
rates were computed. The treatment means from each site mg/kg for Baaga, and 37.75 mg/kg for Gonzales. The
were tested for normality using the Shapiro-Wilk W Test.

Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake

Table 1. Physicochemical analyses of Taal Lake waters and sediments from the three sampling sites (October 2006).
STATION Balakilong Baaga Gonzales
Quality Standards
14 3 16 N, 120 13 56 6 N 14 4 60 N, 121 DENR
Water Parameters
56 33 E 120 56 12 E 7 0 E
Temperature (C) 28.5 28.9 28 26-28
Salinity () 0.07 0.07 0.07 1
Conductivity (S/cm) 1.66 1.68 1.55 1.5-5.00
pH 8.78 8.69 8.69 6.65-8.5
Dissolved Oxygen (ppm) 7.1 6.2 6.5 5
Ammonia (mg/L) nd nd nd 3.18
Cadmium (mg/L) <0.05 <0.05 <0.05 0.01
Lead (mg/L) <0.10 <0.10 <0.10 0.05
Copper (mg/L) 0.07 0.08 0.05 0.05
Quality Standards
Sediment Parameters
pH 6.5 6.5 6.5 6-8.5
Total Ammonia (TAN)(ppm) 404.2 239.1 47.12 100
Unionized Ammonia (U-NH3) (ppm)* 0.73 0.43 0.08 0.18
Cadmium (mg/kg) <1.25 <1.25 <1.25 4.2
Lead (mg/kg) 8.5 11.75 3.5 35
Copper (mg/kg) 80.75 70.00 37.75 <18.7
*computed based on USEPA, 1992 at pH 6.5 and 25C.
DENR (Department of Environment and Natural Resources)
MEEO, OSPARCOM (Ministry of Environment and Energy Ontario, Oslo and Paris Commission)

copper concentration in all sediments is classified as poor showed a significant decrease in the survival rate. The
based on the Oslo and Paris Commissions Criterion on observed reduction in survival rates was much evident
Sediment Quality which was set at 18.7 108 mg/kg. at higher sediment concentrations (75% and 100%) for
both sites.
Water embryotoxicity After 96 h- exposure, significant abnormalities were
As shown in Figure 2, zebrafish embryos exhibited observed in the 50% (p < 0.05), 75% (p < 0.01), and 100%
very high survival rates after 48 h ranging from 82% in (p < 0.01) Baaga sediments relative to the sediment
Balakilong to 91% in the Gonzales site. This is not at all control. Balakilong sediments also exhibited significant
different from the water control (93%). On the other hand, abnormalities at 25% and 50% (p < 0.01), but fewer at
survival in the positive control had been reduced to 30% 75% and 100% concentrations (Figure 5). Deformities and
after 48 h and 12% after 96 h. Abnormalities in the growing abnormalities such as general developmental retardation,
larvae from all sites (water control and exposure groups) underdeveloped eyes and ears, lack of somite formation,
were also minimal and insignificant (data not shown). edemas, unresorbed yolk, and lordosis are shown in Figure
For the hatching success rate, no significant difference 6. As shown in Figure 7, hatching and development were
among the water control (93.3%) and exposure groups delayed in the positive control, Baaga, and Balakilong
(Ban/Bal - 84.4% and Gon-88.9%) was observed. Positive sites. This effect on hatching was already evident starting
control embryos, however, showed highly significant (p < with 25% concentrations for both aquaculture sites (data
0.01) reduction in hatching success rate (13%), relative to not shown). In Gonzales, no significant differences in
negative control and exposure groups (Figure 3). hatching success rates relative to sediment control were
Sediment embryotoxicity
As Figure 4 shows, zebrafish embryos developed
normally in both the Gonzales sediments, regardless of
concentrations, and the sediment (i.e., silica) control after
96 h. However, sediments from Baaga and Balakilong

Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake


Figure 2. Percentage survival of Danio rerio embryos exposed to Taal Lake waters. Only the positive control was found to be
highly different from the water control as indicated by the asterisks (p<0.01). (WC- water control; Gon Gonzales;
Ban Baaga; Bal Balakilong; PC positive control).

Figure 3. Hatching success rate of Danio rerio embryos exposed to Taal Lake waters. Only the embryos exposed to the positive
control showed a highly significant reduction in hatching success rate relative to the water control (**p<0.01).
(WC- water control; PC positive control).

DISCUSSION tawilis (Sardinella tawilis). As such, there is a need to

monitor and assess the suitability and sustainability of
Taal Lake is considered a major aquatic resource in the current aquaculture practices in the area. To effectively
Mega Manila area. The lake supplies nearby towns and do this, improved techniques of environmental toxicity
cities with milkfish (Chanos chanos), tilapia (Tilapia assessment should be integrated in the monitoring.
nilotica), maliputo (Carangoides fulvoguttatus), and

Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake

Figure 4. Percentage survival of Danio rerio embryos exposed to Taal Lake sediments for 96 hours. High survival rate
comparable to sediment control (SC) was shown by the Gonzales sediment. However, Baaga and Balakilong
sediments showed embryotoxic effects to growing embryos especially at the higher concentrations. (*p < 0.05;
**p<0.01). (SC- sediment control; PC positive control).

Figure 5. Percentage abnormalities of Danio rerio embryos exposed to Taal Lake sediments after 96 hours. Asterisks
indicate the sample which significantly differ from the sediment control, *p<0.05 and **p<0.01. (SC- sediment
control; PC positive control).

Chemical monitoring, though useful, can only provide Embryotoxicity of water samples
limited information on the actual or potential biological Based on chemical analysis of the lake water (Table 1), the
activity of contaminants. Biomarker responses, such overall water quality of Taal Lake was maintained at Class
as the developmental endpoints in zebrafish embryos, C status (DENR 1990), making it suitable for fish growth
serve as more precise indicators of the health status and propagation. The outcome of the zebrafish embryo
of the aquatic environment. Biomarkers may serve as assay clearly reflects the below detection limits of the
potential yardstick for chemical pollution since the analyzed chemicals, except copper, in surface waters (Table
intensity of the response may usually indicate the level 1). The survival and developmental events in the zebrafish
or concentration of the chemicals in the extracts of embryos were not arrested nor there have been significant
environmental matrices (Hahn 2002). malformations observed. However, these low mortalities and

Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake

Figure 6 . Embryonic and teratogenic malformations observed in zebrafish embryos exposed to Taal Lake sediments at specified
times. a) normal embryo at 24 h, sediment control; b) somite formation (retarded development) at Baaga (50%);
c) rachischisis (underdeveloped embryo) and failure of tail to detach at Balakilong (25%); d) normal embryo at 48
hours (sediment control); e) edema at Baaga (75%); f) without head, without somites, and with edema at Balakilong
50%; g) normal larvae after 72 hours (sediment control); h) rachischisis, tail not detached, and underdeveloped
eye at Baaga (100%); i) without head, underdeveloped eye, without somites, and edema at Balakilong (75%) j)
brain underdeveloped and eye underdeveloped at Balakilong (75%); k) edema and without somites at Balakilong
(100%); l) edema at Baaga (75%); m) unresorbed yolk and lordosis at Balakilong (25%). B- brain; C cardia;
Ch chorion; P- pigmentation; Ed edema; Eud ear underdeveloped; Eyud eye underdeveloped; Bud brain
underdeveloped; Uys unresorbed yolk sac; HeT helical tail; Tnd tail not detached; WoEy without eyes;
woH without head; woS without somites; Lo lordosis.

Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake

Figure 7. Hatching success rate of Danio rerio embryos exposed to Taal Lake sediments. (100%) after 96 hours and with
extension up to 144 hours.. Baaga and Balakilong sediments showed a highly significant delay in hatching and
a reduction in overall hatching success rate compared to Gonzales and the sediment control. (**p<0.01). (SC
sediment control; PC positive control).

abnormalities, and high hatching success rates in all sites exposed to Taal Lake sediments. The embryos which
do not fully indicate the absence of pollutants in the water were exposed to whole sediments from aquaculture
column. Other unanalyzed pollutants might be present in the sites showed marked and significant developmental
waters at levels below detection, but the level could still be defects. Gonzales, a site of open water fishing, was
high enough to cause embryotoxicity. used as the reference site since no fish pens are found
in this area. Embryos exposed to sediments from this
Although the present study (on the water phase) was not site did not show statistically significant differences
able to demonstrate the capacity of zebrafish embryos to in mortality, abnormality, and hatching success from
discriminate pollution status of waters, we still insist that the control across all dilution levels. The chemical
zebrafish embryos can differentiate between contaminated analyses of the sediments from this site (Table 1)
and uncontaminated sites. Since all sites (reference and showed that the copper and ammonia are within safe
aquaculture sites) have practically similar physicochemical limits (OSPARCOM 1994; MEEO 1993). It is probable
quality, as shown in Table 1, the responses of zebrafish that the levels of harmful chemicals in the site may be
embryos will, therefore, be similar in all sites. In a related too low to have potential developmental effects or there
study by Hallare et al. (2005a), however, zebrafish may have been other unanalyzed contaminants (e.g.,
embryos exposed to diluted water samples from the highly hydrocarbons, pesticides, etc) that may have caused
polluted Navotas River (Philippines) showed pronounced the observed responses.
mortalities and abnormalities. Moreover, undiluted samples
were highly toxic. and resulted in massive coagulation of Balakilong and Baaga were chosen as sampling sites
embryos within a matter of 12 hours. Other studies have also because these are areas of heavy aquaculture and with
documented the ability of growing embryos to discriminate frequent records of fish kills. Significant differences
varying levels of pollution in water systems (Klumpp et al. in survival rates were observed especially at higher
2002; Luckenbach et al. 2003). sediment concentrations (75% and 100%) in both sites.
These results indicate the presence of toxic substances
available at lethal levels. Similarly, Hollert et al. (2003)
Embryotoxicity of sediment samples
demonstrated that the toxic levels of exchangeable metals
A different scenario was shown by our data on sediment
in Eberbach (Neckar River) caused the low survival rates
toxicity. There were sublethal and lethal responses
of zebrafish embryos.
elicited from the developing zebrafish embryos

Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake

Deformities and abnormalities such as general from Gonzales in terms of heavy metal content and
developmental retardation, underdeveloped eyes and ammonia. Baaga and Balakilong sediments are of poor
ears, lack of somite formation, edemas, unresorbed quality (OSPARCOM 1994; MEEO 1993). It is possible
yolk, and lordosis were found to be significant in some that ammonia, copper, and even lead, either individually
sediment concentrations from aquaculture sites. This or in concert, are responsible for the embryotoxicity of the
indicates the presence of toxic particle-bound substances sediments. A gradient of toxicity also exists in the three
which are exerting teratogenic effects (Keiter et al. sites: greatest toxicity potential was found in Balakilong,
2006). Similar observations were reported by Hollert consistent with the highest density of fish cages, followed
et al. (2003) on embryos exposed to sediments from by Baaga sediments, and in Gonzales sediments with
Forellenbach in Neckar River, which receives discharges almost negligible toxicities.
of hospital wastewater and exhibited malformations in
zebrafish embryos. Similarly, heavy metals and perylene
Ammonia and heavy metals as possible toxicants
in the Laguna lake sediments have been shown to cause
Of all the water quality parameters which affect fish
significant abnormalities on fish embryos in 100% native
culture, ammonia is the most important one after oxygen,
sediment concentrations (Hallare et al. 2005b).
especially in intensive systems. Due to exhaustive
Despite the absence of concentration-response relationship aquaculture activities, the primary source of ammonia
in Balakilong sediments, the levels of abnormality in all for Balakilong and Baaga sediments is practically
concentrations were still above that of sediment control. fish feeds. De la Vega (2001) documented that a large
This absence of typical concentration-response relationship amount of unconsumed feeds end up in the bottom of
could be explained by inherent inter-individual variation in the lake. Bacterial decomposition of organic material
susceptibility among embryos exposed to varying doses of such as unconsumed feeds release ammonia into the
the contaminants. Another plausible explanation could be lake waters. According to the Ministry of Environment
the presence of more humic substances in the Balakilong and Energy of Ontario (1993), the Probable Effect Level
sediments. These substances could mask the toxicity of for aquatic life in sediments is 100 ppm total ammonia
the non-polar contaminants. Also, there are studies which concentration. From the chemical data, we can see that
showed that bioaccumulation rate decreases in higher ammonia concentrations in sediments were 239.1 ppm
toxicant concentrations in sediment (e.g., Sormunen et and 404.2 ppm for Baaga and Balakilong, respectively.
al. 2008). These ammonia concentrations exceeded the criterion and
could potentially exert toxic effects.
The reduction in overall hatching success in embryos
exposed to Baaga and Balakilong sediments suggests The unionized form of ammonia (U-NH 3) receives
that Taal Lake sediments have higher teratogenic impact much attention since it has been shown to exert an
than that of Laguna Lake sediments (the largest lake inhibitory effect on embryo development. Lepomis
in the Philippines and found in the same region as Taal cyanellus development was inhibited at 0.49 mg/L
Lake), which did not significantly alter hatching rates U-NH3 (McCormick et al. 1984), which is about the
(Hallare et al. 2005b). Embryos exposed to sediment same concentration as those found in Baaga sediments.
samples showed a 48-h delay to hatching relative to those Samples from Balakilong contained 0.73 mg/kg U-NH3,
of the control embryos, and that retarded embryos tend to a plausible explanation for the elevated embryotoxicity
have low survival rates. These findings coincide with the in embryos exposed to its sediments. More sensitive fish
study of Keiter et al. (2006), where embryos exposed to species such as Salmo gairdneri were found to succumb
Ehingen, Danube River sediments were arrested at the 24-h to much smaller Lowest Observed Effect Concentrations
stage and have not shown further changes. Development (LOEC) value of 0.027 mg/L U-NH3 (Solbe and Shurben
resumed only after 96 h and eventually hatched after 144 1989). This implies that Baaga and Balakilong sediments
h. However, most of the hatched larvae suffered from huge can induce even more pronounced toxicity owing to
edemas. According to Weis and Weis (1987), developmental their higher ammonia levels. This statement, though, has
retardations allow teratogens to act for longer times during to be examined as different species have been shown
critical stages of development, and thus enhance the severity to have different thresholds for sensitivity to ammonia
of the abnormalities produced. (Fairchild et al. 2005; McDonald 2005). So far, there
are no previous data available on the threshold value
What accounts for the contrasting results in embryos for zebrafish embryos. Some researchers suggested that
exposed to Gonzales site and those exposed to heavy unionized ammonia is able to pass through the protective
aquaculture sites is the difference in the chemical quality chorion of embryos (Peterson 1984; Eddy et al. 1990).
of the sites. The two aquaculture sites differ significantly When excess ammonia penetrates the developing tissues

Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake

of the fish embryo, it becomes detrimental to cell-to- at the bottom of the lakes (Dela Vega 2001; Mendiguchua
cell signaling and inhibits the formation of the central et al. 2006). For deep lakes like Taal (average depth of
nervous system (Finn 2007); and this could explain why 24 m), the sediments are protected from water and wind
some embryos in the present study lacked heads and had movement and dilution effect (Vista et al. 2006), unlike
underdeveloped brains. Laguna Lake which is shallow with an average depth of
2.5 m (Nauta et al. 2003). Increased water levels were
Ammonia embryotoxicity could be associated with the shown to prevent waves and currents from reaching the
formation of excessive glutamate that leads to: 1) depletion lakebed and from resuspending sediments; thus, allowing
of ATP and impairment of mitochondrial function and contaminants to accumulate undisturbed (Bachmann et
calcium homeostasis (Felipo et al. 1994; Kosenko et al. al. 2000).
1999; Monfort et al. 2002), 2) inhibition of ammonia
excretion leading to neurological and cytological failure However, natural events such as periodic storms and flooding,
(Ruffer et al. 1981; Monfort et al. 2002) and 3) degradation tidal actions, and water overturns can result in resuspension
of microtubular-associated proteins that causes the alteration of sediment contaminants (Davis 1987; Hollert et al. 2000).
of the microtubular network (Monfort et al. 2002). This resuspension of contaminants could be one possible
explanation for the fishkills in Taal Lake that occur during
Besides ammonia, zebrafish embryos have also been overturns and after storms. Likewise, the role of mobilization
shown to take up heavy metals through facilitated diffusion of dredged sediments has been shown to play a part in
(Li et al. 2004). The levels of copper in the sediments of the episodic fishkills in the Dun River (Neal et al. 2006).
Baaga and Balakilong were high compared with the Resuspension is further complicated by varying patterns of
criterion of the OSPARCOM (1994). Copper has been sulfur upwelling, DO, CO2 concentration, and eutrophication
shown to inhibit hatching success (Hallare et al. 2005b) that occur in the lake ecosystem (Vista et al. 2006).
and to induce lethality (Dave and Xiu 1991) in zebrafish
embryos. The concentrations of lead in aquaculture sites
were at least two-fold higher than in Gonzales, and this can
possibly explain some of the malformations exhibited by
the embryos exposed to Baaga and Balakilong sediments.
Several abnormalities observed in this study have been The main objective of this study was to assess the impact
shown to be induced by lead exposure such as cardiac of fish cage culture on the quality of water and sediments
malformations and edema (Ozoh 1980a), spinal curvatures of Taal Lake, Philippines using developmental biomarkers
(Newsome and Piron 1982), and decreased pigmentation in zebrafish embryos. Results of the bioassay clearly
(Ozoh 1980b). showed that embryotoxic potential is associated with
aquaculture sites and that fish cage culture has a negative
It is highly probable that other metal ions and persistent impact on lake quality. Although the results of this
organic pollutants (POPs) may be implicated to the experiment indicated satisfactory water quality, sediments
observed effects (Strmac et al. 2002; Hallare et al. 2005b), from aquaculture sites were shown to be polluted with
but since they were not included in the chemical analysis, teratogens. This suggests a differential quality between
their potential effects cannot be assessed. Therefore, a next- Taal Lake surface waters and underlying sediments.
level approach characterized by the use of bioassay-directed The water is suitable for fisheries; however, sediments
fractionation is recommended to specifically pinpoint the particularly those in Balakilong and Baaga pose a risk to
toxicants that are responsible for the embryotoxic effects. the early life stages of Danio rerio embryos. The present
study clearly documents that the 96-hour Zebrafish Early
Differential quality of surface water and Life Stage Test is a practical and sensitive bioassay for
underlying sediments assessing the impact of aquaculture on the water and
In the present study, we have shown that zebrafish embryos sediment quality of Taal Lake.
could reveal the differential quality between the water
phase and sediment phase in the Taal Lake ecosystem.
This is very similar to what Canusso et al. (2000) found
in the Po and Rhine rivers and by Hallare et al. (2005a; b) ACKNOWLEDGMENTS
in the Laguna Lake ecosystems wherein the waters were The authors are grateful to the Bureau of Fisheries and
unpolluted yet the sediments were heavily contaminated. Aquatic Resources (BFAR, Philippines) for the permission
The disparity between the two phases might be because to conduct the study, for the site recommendation, and
sediments are sinks of toxic materials (Braunbeck et al. for the provision of additional background data for this
2005) and wastes originating from the aquaculture settle study. We are also truly indebted to the two anonymous

Philippine Journal of Science Hallare et al.: Assessing the Impact of
Vol. 138 No. 1, June 2009 Fish Cage Culture on Taal Lake

reviewers for their very useful and detailed comments on DELA VEGA JT. 2001 Feeds and Feeding Management
an earlier version of this paper. of Tilapia in Cages. Paper Presented at the 4th Southern
Luzon Zonal R and D Review, DAP Tagaytay City,
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