Towards integrated management of a shallow tropical lake: assessment of water quality, sediment geochemistry, and phytoplankton diversity in Lake Palakpakin, Philippines.

The limited carrying capacities of shallow tropical lakes render them more vulnerable to ecological problems like eutrophication. Unregulated human activities such as unsustainable aquaculture and urbanization can alter ecosystem dynamics rapidly, and this warrants more comprehensive researches than what has been previously conducted. Here, we presented an integrated assessment of the nutrient dynamics, phytoplankton diversity, and sediment geochemistry in Lake Palakpakin, a shallow tropical lake of volcanic origin, to understand its deteriorating ecological state. Water, phytoplankton, and sediment samples were collected, and in situ water quality measurements were done during wet and dry seasons in four critical areas in the lake, namely, the inlet, center, sanctuary, and outlet. Results revealed that high light extinction coefficient (1.13 m-1), high turbidity (28 NTU), high phosphate concentration (> 2.0.5 mg/L), and the abundance of Microcystis aeruginosa, Anabaena helicoidea, and Lyngbya sp. indicate that from a relatively healthy lake in 2008, Lake Palakpakin has become a eutrophic to hypereutrophic freshwater body. High concentrations of available nutrients such as N and P were detected in the center and sanctuary sediments, which drive the internal nutrient loading in the lake. We recommend that management efforts be directed towards a whole-ecosystem approach in addressing the problem of eutrophication, especially in shallow tropical lakes.

Organochlorine pesticide residues in surface water and groundwater along Pampanga River, Philippines.

Pesticide use in developing countries such as the Philippines has significantly increased food production. However, the improper and poorly regulated practice of pesticide use may lead to pollution of water resources. To detect and assess the extent of pesticide contamination, residues of organochlorine pesticides were tested in surface water and groundwater in selected areas along the Pampanga River, Philippines. The physicochemical properties of the surface water and ground water were also analyzed and results revealed that phosphate concentrations in surface water and groundwater samples were two to three times higher than the regulatory limits of 0.5 mg L-1, whereas the nitrate concentrations were below the regulatory limit of 7 mg L-1. Results further revealed that surface water and groundwater showed the presence of seven organochlorine pesticides and residues listed in the Stockholm Convention list of 2009 such as dieldrin, endrin aldehyde, α-BHC, ß-BHC, δ-BHC, γ-chlordane, and endosulfan II. The concentrations of organochlorine pesticides including endrin aldehyde, total BHCs (i.e., α-BHC, ß-BHC, δ-BHC), and heptachlor in groundwater were also found to exceed regulatory limits, indicating that these chemicals are still being used illegally and remains a major environmental concern despite the bans and restrictions. We suggest that routine chemical monitoring (including seasonal variations) coupled with biological monitoring using a battery of biomarker tests of organochlorine pesticide and residues along the Pampanga River is necessary to provide inputs for the control and reduction of environmental pollution and for minimizing human health risks.

Assessment of Jatropha curcas L. biodiesel seed cake toxicity using the zebrafish (Danio rerio) embryo toxicity (ZFET) test.

Consequent to the growing demand for alternative sources of energy, the seeds from Jatropha curcas remain to be the favorite for biodiesel production. However, a significant volume of the residual organic mass (seed cake) is produced during the extraction process, which raises concerns on safe waste disposal. In the present study, we assessed the toxicity of J. curcas seed cake using the zebrafish (Danio rerio) embryotoxicity test. Within 1-h post-fertilization (hpf), the fertilized eggs were exposed to five mass concentrations of J. curcas seed cake and were followed through 24, 48, and 72 hpf. Toxicity was evaluated based on lethal endpoints induced on zebrafish embryos namely egg coagulation, non-formation of somites, and non-detachment of tail. The lowest concentration tested, 1 g/L, was not able to elicit toxicity on embryos whereas 100 % mortality (based also on lethal endpoints) was recorded at the highest concentration at 2.15 g/L. The computed LC50 for the J. curcas seed cake was 1.61 g/L. No further increase in mortality was observed in the succeeding time points (48 and 72 hpf) indicating that J. curcas seed cake exerted acute toxicity on zebrafish embryos. Sublethal endpoints (yolk sac and pericardial edema) were noted at 72 hpf in zebrafish embryos exposed to higher concentrations. The observed lethal endpoints induced on zebrafish embryos were discussed in relation to the active principles, notably, phorbol esters that have remained in the seed cake even after extraction.

Toxicity, dioxin-like activities, and endocrine effects of DDT metabolites--DDA, DDMU, DDMS, and DDCN.

BACKGROUND, AIM, AND SCOPE: 2,2-bis(chlorophenyl)-1,1,1-trichloroethane (DDT) metabolites, other than those routinely measured [i.e., 2,2-bis(chlorophenyl)-1,1-dichloroethylene (DDE) and 2,2-bis(chlorophenyl)-1,1-dichloroethane (DDD)], have recently been detected in elevated concentrations not only in the surface water of Teltow Canal, Berlin, but also in sediment samples from Elbe tributaries (e.g., Mulde and Havel/Spree). This was paralleled by recent reports that multiple other metabolites could emerge from the degradation of parent DDT by naturally occurring organisms or by interaction with some heavy metals. Nevertheless, only very few data on the biological activities of these metabolites are available to date. The objective of this communication is to evaluate, for the first time, the cytotoxicity, dioxin-like activity, and estrogenicity of the least-studied DDT metabolites. METHODS: Four DDT metabolites, p,p'-2,2-bis(chlorophenyl)-1-chloroethylene (DDMU), p,p'-2,2-bis(chlorophenyl)-1-chloroethane (DDMS), p,p'-2,2-bis(4-ch1oropheny1)acetonitrile (DDCN), and p,p'-2,2-bis(chlorophenyl)acetic acid (DDA), were selected based on their presence in environmental samples in Germany such as in sediments from the Mulde River and Teltow Canal. O,p'-DDT was used as reference in all assays. Cytotoxicity was measured by neutral red retention with the permanent cell line RTG-2 of rainbow trout (Oncorhynchus mykiss). Dioxin-like activity was determined using the 7-ethoxyresorufin-O-deetylase assay. The estrogenic potential was tested in a dot blot/RNAse protection-assay with primary hepatocytes from male rainbow trout (O. mykiss) and in a yeast estrogen screen (YES) assay. RESULTS: All DDT metabolites tested revealed a clear dose-response relationship for cytotoxicity in RTG-2 cells, but no dioxin-like activities with RTL-W1 cells. The dot blot/RNAse protection-assay demonstrated that the highest non-toxic concentrations of these DDT metabolites (50 µM) had vitellogenin-induction potentials comparable to the positive control (1 nM 17ß-estradiol). The estrogenic activities could be ranked as o,p'-DDT > p,p'-DDMS > p,p'-DDMU > p,p'-DDCN. In contrast, p,p'-DDA showed a moderate anti-estrogenic effect. In the YES assay, besides the reference o,p'-DDT, p,p'-DDMS and p,p'-DDMU displayed dose-dependent estrogenic potentials, whereas p,p'-DDCN and p,p'-DDA did not show any estrogenic potential. DISCUSSION: The reference toxicant o,p'-DDT displayed a similar spectrum of estrogenic activities similar to 17ß-estradiol, however, with a lower potency. Both p,p'-DDMS and p,p'-DDMU were also shown to have dose-dependent estrogenic potentials, which were much lower than the reference o,p'-DDT, in both the vitellogenin and YES bioassays. Interestingly, p,p'-DDA did not show estrogenic activity but rather displayed a tendency towards anti-estrogenic activity by inhibiting the estrogenic effect of 17ß-estradiol. The results also showed that the p,p'-metabolites DDMU, DDMS, DDCN, and DDA do not show any dioxin-like activities in RTL-W1 cells, thus resembling the major DDT metabolites DDD and DDE. CONCLUSIONS: All the DDT metabolites tested did not exhibit dioxin-like activities in RTL-W1 cells, but show cytotoxic and estrogenic activities. Based on the results of the in vitro assays used in our study and on the reported concentrations of DDT metabolites in contaminated sediments, such substances could, in the future, pose interference with the normal reproductive and endocrine functions in various organisms exposed to these chemicals. Consequently, there is an urgent need to examine more comprehensively the risk of environmental concentrations of the investigated DDT metabolites using in vivo studies. However, this should be paralleled also by periodic evaluation and monitoring of the current levels of the DDT metabolites in environmental matrices. RECOMMENDATIONS AND PERSPECTIVES: Our results clearly point out the need to integrate the potential ecotoxicological risks associated with the "neglected" p,p'-DDT metabolites. For instance, these DDT metabolites should be integrated into sediment risk assessment initiatives in contaminated areas. One major challenge would be the identification of baseline data for such risk assessment. Further studies are also warranted to determine possible additive, synergistic, or antagonistic effects that may interfere with the fundamental cytotoxicity and endocrine activities of these metabolites. For a more conclusive assessment of the spectrum of DDT metabolites, additional bioassays are needed to identify potential anti-estrogenic, androgenic, and/or anti-androgenic effects.

DNA damage induced by genotoxicants in zebrafish (Danio rerio) embryos after contact exposure to freeze-dried sediment and sediment extracts from Laguna Lake (The Philippines) as measured by the comet assay.

Laguna Lake, Philippines, is utilised as a resource for drinking and irrigation water and a major source of animal protein for more than 11 million residents. However, it also serves as a huge sink for anthropogenic pollutants from a variety of direct or indirect discharges. Given their well-known properties to accumulate organic pollutants, Lake Laguna sediments have been investigated by use of a newly developed sediment-contact assay to determine the bioavailable fraction of the total genotoxic hazard potential. Freshly fertilized zebrafish eggs (2 h after fertilization) were allowed to develop while exposed to different concentrations of freeze-dried sediments (exposure to bioavailable compounds) as well as organic extracts (complete genotoxic hazard potential including bioaccumulation) prepared from sediments collected at five sites within the lake. Following a 96 h exposure, single-cell suspensions were prepared from macerated larvae and analyzed for DNA strand-breaks by use of the comet assay. Genotoxicity could be identified in both solid-phase exposure scenarios as well as after exposure to organic sediment extracts. Only the South Bay (SB) site did not show a significant genotoxic burden relative to controls in tests of both freeze-dried sediment and organic extracts. In contrast, the Northwest Bay site displayed pronounced genotoxicity to the zebrafish embryos in both exposure scenarios. Perylene and copper in the sediments could be identified as potential causes of the genotoxic response. To compare the results (maximum induction coefficients) of zebrafish embryos with an established comet protocol, rainbow trout liver cells (RTL-W1) were exposed to the same extracts. The findings correlated well (Spearman correlation r=0.90), proving a good reliability of the results from zebrafish primary cells. In conclusion, the present study demonstrates that the bioavailable fraction of the genotoxic pollutants may pose a threat for both benthic organisms and human health via drinking-water and fish consumption.

A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos.

Broad consensus exists that whole-sediment exposure protocols represent the most realistic scenario to simulate in situ exposure conditions. So far, however, several endpoints including genotoxicity in vertebrate-based systems could be tested only after transfer of particle-bound substances into the aqueous phase. The present study was carried out to develop a protocol for generating a suspension of single cells from sediment-exposed zebrafish embryos that is suitable for detecting particle-bound genotoxicity in the alkaline single cell gel electrophoresis (comet assay). In this solid-phase genotoxicity assay, a whole-body cell suspension derived from zebrafish embryos exposed to native (whole) sediments is assayed in the comet assay. Several chemical and mechanical isolation procedures were compared to optimize cell yield and minimize DNA damage by the method itself. If compared to collagenase isolation, mechanical cell dissociation gave less DNA damage; trypsinization resulted in similarly low DNA damage but significantly lower cell yield. In order to test the optimized protocol, effects of well-known genotoxicants (4-nitroquinoline-N-oxide, nitrofurantoin, hydrogen peroxide, benzo[a]pyrene) and of two sediments from the upper Rhine River (Germany) on zebrafish embryos were investigated. Results documented clear-cut genotoxicity for all four substances and for one of the two whole-sediment samples. An ultraviolet (UV) light exposure of whole embryos and primary cultures from embryos elucidated only minor effects for the whole embryos compared to the primary cells. Consequently, UV irradiation cannot be suggested as a positive control in intact zebrafish embryos. In conclusion, the newly developed sediment contact assay can be recommended for the detection of both single substances but also the bioavailable fraction of the total hazard potential of sediments.