self-evaluation of the project … · toward establishment of micro-organism method ... study is...

1

The 4th Meeting of SEAFDEC Program Review for Japanese Trust Fund V,

and Identification of Prioritized Issues for Future Regional Program Formulation 25-26 February 2014, Imperial Queen's Park Hotel, Bangkok, Thailand

WP03.1b

SELF-EVALUATION OF THE PROJECT IMPLEMENTATION

Project Title: Food Safety of Aquaculture Products in Southeast Asia Lead Department: Aquaculture Department Lead Country: Philippines Total Duration: 2010-2014 Total Budget: 137,047 US$

. Year Budget (US$) . 2010 35,100 2011 20,783 2012 42,064 2013 39,100

Evaluation period: 2010-2013 1. INTRODUCTION The use of antibiotics and other chemicals in aquaculture is widely practiced to help meet the increasing demand for aquaculture food. These antibiotics and chemicals detected in aquaculture products appear to derive from material inputs during rearing, mostly from contaminated feed ingredients and therapeutants for prevention or treatment of diseases. Thus, cultured shrimps and fish in various stages from hatcheries to grow-out ponds are exposed to chemical contamination. On the other hand, with the ever-growing and worldwide concern for food safety, fish farmers are faced with the challenge of producing safe food from farm to fork. There are very limited data available on the withdrawal period of antibiotics and the presence of chemical residues in aquaculture products from the region. Considering the growing-awareness on issues of food safety of aquaculture products, it is an urgent matter that SEAFDEC should take the lead in establishing regional guidelines on the right usage of antibiotics and other chemical inputs that will allow farmers to increase production of safe food using the environment-friendly technologies. 2. PROJECT 2.1 Objectives This Project “Food Safety of Aquaculture Products in Southeast Asia” is being proposed to: 1) Contribute in the establishment of guidelines on the production of safe aquaculture products from

Southeast Asia; 2) Determine the presence and levels of commonly used chemicals in aquaculture in aquaculture

products such as fish and shrimps; 3) Compile and disseminate SEAFDEC guidelines on the use of antibiotics and chemicals in

aquaculture to the ASEAN region; and 4) Implement training course/workshop to promote food safety awareness in the ASEAN region.

2.2 Project description The Aquaculture Department of SEAFDEC has been managing and coordinating all project activities. Other ASEAN member countries which were identified as core countries in the project have also been involved in implementing the relevant activities.

2

Surveillance activity of chemical contaminants such as pesticides, mycotoxins and antibiotics will be continued based on the results of TF4. Particularly, the withdrawal period of the antibiotics for tropical aquatic species has not been studied enough to ensure the safety for human consumption, compared to the temperate species. Antibiotics are used in aquaculture to prevent and treat diseases that affect farmed shrimp and fish. The indiscriminate use of antibiotics could lead to drug resistant strain and multiple antibiotic resistances in bacteria. From the viewpoint of food safety of aquaculture products, there is a pressing need to survey the chemicals used in aquaculture at present. High performance liquid chromatography (HPLC) is the method of choice for the routine analysis of antibiotic detection. However, antibiotic detection using HPLC requires equipment that is not affordable to small laboratories; and skilled chemists/technicians are needed to do the analysis. Therefore, an alternative method of antibiotic detection that is easy and cost effective is needed. The expected outputs for the project will include the establishment of guidelines on appropriate administration and withdrawal of chemicals in collaboration with ASEAN. The guidelines will be utilized for some possible action or policy formulations by governments of the ASEAN member countries. Seminars and lectures on food safety awareness for stakeholders will also be conducted. All the activities/sub-activities involved in this project are in line with the Resolution and Plan of Action, which were endorsed in ASEAN-SEAFDEC Conference on Sustainable Fisheries for Food Security Towards 2020 held at Bangkok in June 2011. Project implementation and the major outputs I. Withdrawal periods of antibiotics in some aquatic species cultured in the tropics

The withdrawal periods of oxytetracycline (OTC) and oxolinic acid (OXA) in milkfish Chanos chanos, hybrid red tilapia Oreochromis niloticus ×O. mossambicus, mangrove red snapper Lutjanus argentimaculatus, and orange spotted grouper Epinephelus coioides, were determined in a tropical culture environment (Table 1).

Table 1. Withdrawal periods (day) of oxytetracycline (OTC) and oxolinic acid (OXA) in tropical culture species

Milkfish Hybrid red tilapia

Mangrove red snapper

Orange-spotted grouper

Temp. °C 28.7-31.0 28.0-31.0 26.5-29.5 25.0-29.5 OTC 22 26 21 21 OXA 27 17 18 17

A. location of the project: – Philippines

B. perceived benefits: – Knowledge of withdrawal periods of antibiotics in some aquatic species

C. target beneficiaries: – National government, LGUs, academes, fish-farmers

1) Are the activities happening as planned? – Yes.

2) Is the project reaching the target beneficiaries? – Results are available whenever there are interested parties.

3) Is the project reaching the intended number of participants? – N/A

4) How satisfied are the participants/target groups of the project? – N/A

5) How should the planned activities be modified to work better? – N/A

6) What lessons have we learned so far? – The results of the study will help in the establishment of appropriate withdrawal periods of

3

antibiotics in studied fish species in a tropical culture environment, with the fish farmers as the stakeholders of the established data.

7) Do activities meet the needs of participants/stakeholders of the project? – Yes.

II. Surveillance of chemical contaminants in aquaculture products and feeds Ø A total of 69 aquaculture products from the three regions of the Philippines were obtained and

processed to determine residues of two antibiotics, OTC and OXA, and 18-20 Organo-Chlorine Pesticides (OCPs). OXA was commonly used compared with OTC with one sample exceeded the PEL (Permissible Exposure Limits) and MRL (Maximum Residue Limits). The most common OCP detected in aquaculture products was Methoxychlor which was detected in samples from the three regions (Luzon, Visayas, and Mindanao). Since the surveillance was finished in the end of 2012, the terminal report was attached in Appendix I.

Ø Toward establishment of micro-organism method to detect antibiotecs, no output yet. The study is laboratory-based. A micro-organism method of oxytetracycline and oxolinic acid detection will be developed that will benefit laboratories that do not have HPLC, the approved method of antibiotic detection

A. location of the project: – Philippines

B. perceived benefits: – Knowledge of chemical contaminants in aquaculture products and feeds – Simple methodologies on detection for antibiotics

C. target beneficiaries: – National government, LGUs, academes, fish-farmers

1) Are the activities happening as planned? – For surveillance: Yes. – For micro-organism method: No, delay in acquiring needed media and organisms.

2) Is the project reaching the target beneficiaries? – Results are available whenever there are interested parties.




6) What lessons have we learned so far? – The antibiotic oxolinic acid was commonly used compared with oxytetracycline. – The most common organochlorine pesticides detected in aquaculture products was

methoxychlor. 7) Do activities meet the needs of participants/stakeholders of the project?

– Yes. III. Establishment of guidelines for the appropriate administration and regulation of chemical use and technology extension Ø In investigating the status of chemical usage and regulations in Southeast Asia, SEAFDEC AQD,

through this GOJ-TF5 supported study, is collaborating with DOF Malaysia in harmonizing the guidelines for chemical use in aquaculture and measures to eliminate the use of harmful chemicals in the ASEAN. Three regional workshops were organized from 2009-2012 which resulted in a draft document GUIDELINES FOR THE USE OF CHEMICALS IN AQUACULTURE AND MEASURES TO ELIMINATE THE USE OF HARMFUL CHEMICALS being circulated in the ASEAN. Once the ASEAN guidelines on chemical use in aquaculture is finalized and circulated among the ASEAN Member Countries, corresponding guidelines in chemical use will be formulated and circulated among SEAFDEC Member Countries.

Ø The 2013 semi-annual progress meeting discussed on this and suggested that the guideline should include the detailed recommendation for antibiotics/chemicals usage, which were not included in

4

the ASEAN guideline draft. In addition, emerging chemicals concerned recently among the Southeast Asian region such as ethoxyquin should be included as well as information on withdrawal periods for antibiotics.

A. location of the project: – Done in the Philippines, but the information obtained is from all member countries

B. perceived benefits: – Knowledge of the appropriate administration and regulation of chemical use

C. target beneficiaries: – National governments, LGUs, academes, and fish-farmers in all member countries

1) Are the activities happening as planned? – No

2) Is the project reaching the target beneficiaries? – Not yet. However, the information would be significance once established.




6) What lessons have we learned so far? – ASEAN guideline does not include the information of withdrawal period of the antibiotics.

7) Do activities meet the needs of participants/stakeholders of the project? – Yes

International seminar on Food Safety in Aquaculture in Southeast Asia Ø The seminar was held on 22 January 2011 at the Training and Information Division (TID)

Conference Room, SEAFDEC/AQD, Tigbauan 5021, Iloilo, Philippines. Ø Total of 47 Participants from SEAFDEC Member Countries participated (Cambodia 1, Malaysia 1,

Myanmar 1, Philippines 39, Singapore 3, and Thailand 2). Ø The objectives of the seminar were to a) review and analyze the new information on the promotion

food safety in aquaculture practiced in Southeast Asia, and b) discuss the correct way and tackle immediate action to address the issues on food safety in the region.

Ø The following topics were presented and discussed. 1) Good aquaculture practices: Current knowledge and future priorities 2) Fish health, nutrition & food safety in aquaculture 3) Hazard Analysis Critical Control Point (HACCP) – principles, practices & guidelines 4)Technological tools:

– Biological evaluation-Microbial Analysis in aquaculture – Chemical and physical evaluation 1 – heavy metal analysis in fish – Chemical and physical evaluation 2 – antibiotic residue analysis – Biotechnology tools – detection of food borne pathogens using PCR-based methods

5) Food safety management systems – policy and regulations 6) Food safety program in aquaculture: training and information needs and strategies

Ø The following recommendations were obtained. 1) Shared responsibility among the government sector, producers, processors and consumers 2) Reinforce the “farm to table” approach 3) Harmonize the common policy on food safety in Southeast Asia

Ø Challenges that face the implementation of food safety in aquaculture in SEA were summarized as follow. 1) Problem is more on funding, the need for capital 2) Educating the farmers 3) Needs time for implementation 4) As of now certification is voluntary: mandatory for exporting farmers only; but planning to

make it mandatory later on. A. location of the project:

5

– SEAFDEC/AQD, Phillipines B. perceived benefits:

– Knowledge and technologies necessary to promote food safety of aquaculture productions C. target beneficiaries:

– National governments, researchers and/or academes 1) Are the activities happening as planned?

– Yes 2) Is the project reaching the target beneficiaries?

– Yes 3) Is the project reaching the intended number of participants?

– Yes 4) How satisfied are the participants/target groups of the project?

– Highly satisfied. 5) How should the planned activities be modified to work better?

– N/A 6) What lessons have we learned so far?

– Effort to dissemination of knowledge and technology 7) Do activities meet the needs of participants/stakeholders of the project?

– Yes Training Course on Food Safety of Aquaculture Products

Year Date No. of Participants/Gender Ages (Yr) Countries represented

2012 12 – 16 November

14 participants (4F, 10M)

24-59 Bangladesh (9) Cambodia (1*) Philippines (3) Thailand (1*)

* GOJ-TF Funded A. location of the project:

– AQD and University of the Phillipines Visayas; Processing Plant Site in the Phillipines B. perceived benefits:

– The participants are expected to acquire knowledge and technologies necessary to identify appropriate considerations for good aquaculture practices applicable in their situation, as well as methodologies for proper surveillance. It will also increase their awareness and commitment to promote wholesome and safe aquaculture products.

C. target beneficiaries: – Aquaculturists, extension workers, fish farmers, researchers and/or seafood processors

1) Are the activities happening as planned? – Yes, the schedule was followed.

2) Is the project reaching the target beneficiaries? – Yes.

3) Is the project reaching the intended number of participants? – Due to budget constraint, the GOJ-TF slots were opened for 3-4 selected member countries only.

4) How satisfied are the participants/target groups of the project? – The trainees commended the implementation of the course as excellent in all aspects.

5) How should the planned activities be modified to work better? – Longer duration of training. To have more practical sessions and field visit particularly on

HACCP and GAP/BAP topics. 6) What lessons have we learned so far?

– Constant communication and consultation with the resource persons is important. The comments and suggestions of the participants are always given consideration during preparatory meeting.

7) Do activities meet the needs of participants/stakeholders of the project? – Yes, based on the high-rating of their evaluations.

Major outputs:

6

– Knowledge and technologies extension, which are necessary to identify appropriate considerations for good aquaculture practices

IV. Others International Workshop on Fish Health Management: Accelerating Awareness and Capacity-Building in Southeast Asia Ø International workshop on food safety of aquaculture products in Southeast Asia was held at Iloilo

City, Philippines on 8-9 May 2013. Ø A total of 150 aquaculture and food safety experts, scientists, representatives and observers from

11 countries attended. Ø The workshop aimed to: 1) acquire knowledge, technologies and recommendations that are

necessary for SEAFDEC Member Countries to integrate into their food safety and traceability standards setting process involving aquaculture products in their respective situations; and (ii) to increase the Member Countries’ awareness and commitment to promote wholesome and safe aquaculture products.

Ø In the Workshop Discussion, research gaps, capacity-building, and regulatory, harmonizing policies among member countries were discussed.

Ø The details of the workshop discussion were described in the workshop report together with the book of abstracts, which are available from the SEAFDEC/AQD homepage.

Ø After the workshop, the results of evaluation of the workshop which was evaluated by the participants were obtained as follows. 1) Relevance, appropriateness and importance of topics presented to the objectives of the

workshop: mostly gave a rating of “very good” and no “poor”. 2) Speakers’ quality of presentation: mostly gave a rating of “very good” and no “poor”. 3) Venue: mostly gave a rating of “very good” and no “poor”. 4) Food: mostly gave a rating of “very good” and no “poor”. 5) Organizers: majority gave a highest rating of “excellent” and “very good” with no “poor”. 6) Workshop as a whole: mostly gave a rating of “very good” and no “poor”.

A. location of the project: – Iloilo City, Philippines

B. perceived benefits: – Knowledge on gaps on food safety of aquaculture products in the Southeast Asia and ideas on its

countermeasure C. target beneficiaries:

– Fish and shrimp farmers, academes, LGU and national government officers 1) Are the activities happening as planned?

– Yes. 2) Is the project reaching the target beneficiaries?

– Yes. 3) Is the project reaching the intended number of participants?

– Yes. 4) How satisfied are the participants/target groups of the project?

– Highly satisfied. 5) How should the planned activities be modified to work better?

– Earlier invitation for potential presenters 6) What lessons have we learned so far?

– Importance of information dissemination and dialogue with private sector/ fish and shrimp farmers, LGU officers.

7) Do activities meet the needs of participants/stakeholders of the project? – Yes.

3. SUMMARY OF ACTIVITIES AND ACHEIVEMENTS OF THE PROJECT

3.1 Summary of Activities and Budget Used:

7

Activities from 2010 – 2013 Type * Budget Spent Beneficiaries /Country

Year 1: 2010

• Withdrawal period of antibiotics 1 8,991.60 Aquafarmers/ All MCs

• Surveillance of chemical contaminants 1 6,122.35 Aquafarmers/ All MCs

• Establishment of guideline and Tech. Exten. 1&2 0 Aquafarmers/ All MCs

• Others (publication, Annual progress meeting/ workshop, etc.) 5 1,542.55 Aquafarmers/

All MCs Year 2: 2011



• Establishment of guideline and Tech. Exten. 1&2 8,459.34 Aquafarmers/ All MCs


All MCs Year 3: 2012



• Establishment of guideline and Tech. Exten. 1&2 3,646.39 Aquafarmers/ All MCs


All MCs Year 4: 2013 Jan-Jun



• Establishment of guideline and Tech. Exten. 1&2 0 Aquafarmers/ All MCs


All MCs * Type (Types of Activities): 1) Research activities, 2) Training and capacity building, 3) Technical development and improvement activities, 4) Policy development activities, 5) Information activities

3.2 Overall Project Achievements Achievement of the project objectives and the major outputs The project goal of establishing and disseminating guidelines on safe aquaculture products and on use of antibiotics and chemicals is very relevant to the needs of the aquaculture industry in the ASEAN region. This issue is very relevant now with increasing population which requires intensification of fish production to produce more food. Outcomes (Data) of some studies seem to be very good. The study leaders should try to publish the results obtained in the program. This is because the guideline is expected to be made based on the authorized data. 3.3 Evaluation of the project outcomes

8

3.3.1 Effectiveness : The completion of the Guidelines on Chemical use in aquaculture would be the most significant contribution of the Program to the development of sustainable fisheries in the ASEAN region. To what extent were the objectives achieved/are likely to be achieved?

Around 70%. The progress of implementation of the project should be hurried. What are the major factors influencing the achievement or non-achievement of the objectives?

Procurement of the experimental materials takes plenty of time. It is the major factor.

3.3.2 Efficiency :

Depending on the activity, the efficiency differed. Were activities cost-efficient?

No. Particularly, the surveillance of chemicals should have been implemented in more cost-efficient manner. Were objectives achieved in time? Partially, Yes. However, some activities are behind the schedule. What could have been done differently to complete the project more effectively? The surveillance of chemicals in aquaculture products should have been done using samples collected from commercial markets. If done so, it would be more efficient and comprehensive. Organo-Chlorine Pesticides (OCPs) are more toxic to shrimps than to human beings and the origin of OCPs which were detected in shrimps should have been examined.

3.3.3 Sustainability :

In order to increase the aquaculture products, SEAFDEC should take the leadership in establishing regional guidelines on the right usage of antibiotics. To what extent did the benefits of a project continue after the funding ceased?

If the project objectives are achieved, the value of the aquaculture products of this area would greatly increase. What were the major factors that influenced the achievement or non-achievement or sustainability of the project? Networking and frequent exchange information on food safety would be the major factors. What lessons have we learned for the future? It is necessary to publish results in the reviewed international scientific journals so that the food safety is approved of all over the world. What would we like to continue?

Active collaboration with other regional and international organization, such as NACA, APFIC, and FAO as well as SEAFDEC Member Countries would be highly recommended.

4. Countries Beneficial All Member Countries are deserved to receive knowledge and technologies on food safety to heighten the sustainable fisheries and aquaculture production in the region.

9

Appendix I

Terminal report on Surveillance of chemical contaminants in Activity 2

Surveillance of chemical contaminants in aquaculture products and feeds: Levels and classification of contaminants in aquaculture products, aquafeeds and aquafeed ingredients in the Philippines and other

Asian countries

M. R. Catacutan, R. M. Coloso and M. Arnaiz SEAFDEC Aquaculture Department, Tigbauan, Iloilo 5021, Philippines

Abstract A total of 69 aquaculture products from the three regions of the Philippines were obtained and processed to determine residues of two antibiotics, Oxytetracycline (OTC) and Oxolinic acid (OXA), and 18-20 Organochlorine Pesticides (OCPs). The antibiotics were assayed by HPLC while for the OCPs were assayed using a gas chromatography with electron capture detector (GC-ECD). Some samples were positive for the antibiotics and OCPs residues and these comprised of low and high value fish species. The assessment of results were based on the values set for TWA (8-hr time weighted average) and PEL (Permissible Exposure Limits) values based on OSHA (Occupational Safety and Health Administration, US based) and MRLs (Maximum Residue Limits, Japan Food Chemical Research). This research work showed that the antibiotic OXA was commonly used compared with OTC with one sample exceeded the PEL and MRL limits (P. vannamei). On the other hand, the most common OCP detected in aquaculture products is Methoxychlor which was detected in samples from the three regions (Luzon, Visayas, and Mindanao). In one sample (Macrobrachium sp.) from Luzon, the levels of the two OCPs (Endrin ketone and Endosulfan I), were found harmful based on PEL and MRL.

Introduction A. Review of Literature

A wide range of chemicals are used in the culture of popular aquaculture species. Indiscriminate use of these chemicals poses dangers to human health as some of these chemicals have been detected in aquaculture products (Londershausen, 2009). A survey in the Philippines (Lacierda et al.,1996) revealed that more than 100 chemical products are used for rearing, prophylaxis, and treatment purposes in shrimp and milkfish culture facilities. The increased usage and the number of these drugs and other chemicals throughout the years have been quite predictable since world aquaculture production and the number of species for culture had been increasing. For health reasons of consumers and the safety regulations imposed by importing countries on aquaculture products, there is a pressing need to survey the chemicals used in aquaculture at present.

One study showed that in the Philippines a significant level of the pesticide, organotin, is in tissues of milkfish samples from brackish water ponds (Coloso & Borlongan, 1999). This pesticide is used by farmers to control the population of water snails in fish ponds. Detecting the presence of these chemicals in aquaculture products is important in the region because in 2004, a manual is published by the Marine Fisheries Research Department of SEAFDEC on “Detection of Antibiotic Residues in Aquaculture Products”. The analytical method in detecting these chemicals is by chromatography and such as the antibiotics, Oxytetracycline and Oxolinic acid, fish tissues (1993 and 2008).

This work is about the survey of chemical contaminants in aquaculture products in the Philippines. This is in line with the promotion of food safety awareness in the region with regards to fish and the like produced through aquaculture. B. Objectives The objectives of this work is to survey the chemical contaminants in aquaculture products and feeds which will be done in the Philippines and in selected lesser developed Asian countries. The

10

aims are the following, 1) to determine levels of commonly used chemicals in aquaculture in aquaculture products such as fish and shrimps in developing ASEAN member countries, and 2) to determine levels of chemical contaminants, such as antibiotics, fungicides, feed additives, toxin binders etc. in aquafeed and feed ingredients, and in aquaculture products. C. Importance of the Study Many chemicals are used in aquaculture production. Some of these chemicals have been banned while some of these are regulated. The uncontrolled use of these chemicals have been reported such that government agencies become stricter because of the adverse effects of these chemicals on human health, the environment and the development of pathogen resistance. Results of this study will be good information on the extent and type of chemicals being used for specific species and also in areas where these chemicals are utilized.

Materials and Methods Sampling

In 2010, samples of aquaculture products such as fish and crustaceans were obtained from either of the following culture systems: ponds, cages or pens. These were obtained from the three major islands of the Philippines (Luzon, Mindanao & Visayas). Live or fresh samples were immediately placed in styrofoam boxes with ice to avoid spoilage. Fresh samples were also obtained from wet markets in some localities visited. Feed samples on the other hand, were placed in labeled plastic bags with air carefully removed. Each sample was carefully labeled and packed and these were transported to the laboratory at SEAFDEC in Tigbauan. Samples were kept in the freezer until processed for the assays of residues of antibiotics and pesticides and heavy metals in feed/feed ingredient samples.

In 2011, only one field sampling was done and this was in February conducted in Palawan province. Samples were obtained from the northern and southern part of the province and also from Puerto Princesa City. Most samples were obtained in sea cages and a few from brackishwater earthen ponds. A total of twelve samples (one was discarded) were obtained in two and a half days of sampling. Samples that were collected consisted mostly of fish species, namely, milkfish (Chanos chanos), grouper (Epinephelus coioides), snapper (Lutjanus argentimaculatus), sea bass (Lates calcarifer) and rabbit fish (Siganus guttatus). Only one crustacean sample was obtained, the shrimp Penaeus monodon.

There was no sampling trip conducted in 2012. However, we obtained aquaculture product samples in other localities during sampling trips for other purposes and we have piggy-backed with other trips of SEAFDEC colleagues. No additional samples of feeds were obtained since the original yearly budget as conceptualized was cut down by a third in the succeeding two years and no reagents for the other chemical analyses. Procedure for Chemical Analyses of Samples

The antibiotics determined in samples were Oxytetracyline (OTC) and Oxolinic acid (OXA). The method for the assay of OTC was by HP Liquid Chromatography with Ultraviolet detection according to Carignan,G. et al (1993), while the assay of OXA was by HPLC according to Borlongan, I.G. & Ng Poh Chuan, J. (2004). For the OTC assay, the extraction of the residue was done using the solvent system of metaphosphoric acid and dichloromethane or DCM. For the OXA, the extraction solvent system was acetonitrile and Na2SO4.

The presence of organochlorine pesticides or OCP in samples was determined by extraction of the residues in samples using acetonitrile and purification with Florisil column chromatography. The purified extract was diluted with n-hexane and assayed by gas chromatography with electron capture detector (GC-ECD). This was a revised method from the original method used at Marine Fisheries Research Department, Singapore (Pesticide Residue Analysis-Gas Chromatograph Mass Spectrometer, 2008).

The standard for the organochlorines showed only 18 peaks in the chromatograph (GC-ECD). The following are the OCPs in the standard with their respective range of retention time (minutes) in parenthesis: alpha-BHC (6.188-6.255), gamma-BHC (Lindane) (6.802-6.885), beta-BHC (6.954-7.035), delta-BHC (7.387-7.475), Heptachlor (7.487-7.572), Aldrin (7.881-7.973), Heptachlor

11

epoxide (isomer B) (8.662-8.761), trans-Chlordane (8.933-9.033), cis-Chlordane (9.139-9.242), 4,4'-DDE (9.178-9.276), Endosulfan I (9.434-9.536), Dieldrin (9.584-9.688), Endrin (10.044-10.154), 4,4'- DDD (10.171-10.279), Endosulfan II (10.294-10.405), 4,4'- DDT (10.575-10.688), Endrin aldehyde (10.691-10.804), Methoxychlor (10.912-11.028), Endosulfan Sulfate (11.735-11.857), and Endrin ketone (12.052-12.179).

In the calculations of OTC and OXA levels, the values derived below the linear regression curve or the Linear Regression Equation of the Antibiotic Standard were considered as nil. For the assessment of organochlorine pesticides (OCs) values, these were based on OSHA (Occupational Safety and Health Administration, US based) and exposure limits levels in OCPs were compared with the PEL (Permissible Exposure Limits) values in ppm TWA (8-hr time weighted average).

Feed and feed ingredients samples were stored for analyses of heavy metals. Confirmatory Test for antibiotics residual analyses (which included OTC and OXA) was done by re-injection of the final extract and re-extraction of the muscle sample. On the other hand, due to limited amount of chemicals and reagents, confirmatory tests for Organochlorine pesticides were done by reinjection only of the final extract using GC-ECD.

Results In 2010, samples of aquatic products totaled 56 which included 10 fish species (48) and 3 crustacean species (8). Although high value species were also obtained which included grouper, red snapper and sea bass, the fish species samples were predominantly milkfish (22) and tilapia (17). Majority of the shrimp samples were from the Visayas area. Of the 56 samples, only 34 were analyzed at the end of 2010 and the rest were in different stages of sample preparation while some these were ready for injection to the HPLC. Method validation for other samples was done. Results showed that two samples from the same locality were positive for OTC and OXA. Feed samples were collected in the sampling area when the owner of the farm would allow us. In one case, we were not allowed to take samples unless we signed an agreement that we will inform them first the results of analysis of their samples. In another case we were requested to inform the owners first the results before making the report. In Luzon, we were told that positive results when known to the media will affect their income since people will stop buying their products. From these observations in the field, the results of our study will be sensitive to some sectors of the aquaculture industry and hence confirmatory runs were done in 2011 and 2012 on samples with positive results. Method validation was also conducted. However, no samples of feed and feed ingredients were analyzed for heavy metal contaminant since there was no available fund. Furthermore, for the determination of other contaminants such as feed additives, toxin binders, fungicides, these were not conducted although samples have already been collected for these purposes. The budget for the study dwindled in the second and third years. Hence, analysis of samples was limited to two antibiotics and 22 organochlorine pesticides. Although it was in the objective of this study, samples were not obtained from other less developed countries as this entails travel and funding support. At the end of 2011, which was 18 months from the start of the study, a total of 70 samples of aquaculture products which comprised of about 13 species have been brought to the laboratory from the provinces in Luzon, Visayas & Mindanao (Figures 1-3). Generally, the samples we got from Palawan were expensive as these were mostly high value species. Results of OTC, OXA and Pesticides assays are in Table 1 for Luzon, Table 2 for Visayas and Table 3 for Mindanao. In Table 4, is a list of chemical contaminants and the minimum residue limits (MRLs) for some fish some species. In our results, fish species found positive for chemical residues were a mixture of low and high value species. In Table 1, a total of 31 samples were obtained which were mostly freshwater species predominantly bangus (10) and tilapia (9). Four samples were not analyzed for antibiotics. The antibiotic OTC was not detected in any of the samples analyzed and only one sample contained OXA which do not have yet the PEL (Permissible Exposure Limit, US) but below the MRL set by Japan (Agricultural Chemicals, Table 4). A fourth of the samples (7 out of 31 samples) contained OCPs but only one sample (Macrobrachium sp.) had exceeded the MRL and contained Endosulfan I, Endrin ketone. The chemical Methoxychlor was found in four out of seven fish samples but below the MRL level of 0.70734 OSHA Exposure Limits in ppm TWA. In milkfish samples only one was positive but

12

the level did not exceed the MRL. The other OCPs detected that were below the MRL were, Heptachlor epoxide isomer B, Endrin, 4,4’-DDT and Trans-Chlordane. In Table 2, a total of 27 samples were obtained and these were mostly bangus and shrimps. Only one sample was positive for OTC and three for OXA, but, these did not exceed the set MRL. The rest of the samples were negative for these antibiotics. For the pesticides, only two samples were found positive. Each of the sample was positive for two OCPs (P. monodon: Endosulfan II and Endosulfan sulfate; bangus: Heptachlor and Methoxychlor). The levels of thes chemicals were below the set limits of MRL. In Table 3, a total of 11 samples were analyzed from Mindanao area, and again, almost half of these were bangus. One bangus sample was positive for OTC, OXA and Methoxychlor and another bangus sample was positive for Aldrin. The shrimp sample (P. vannamei) was positive with OTC which exceeded the maximum residue levels set by both the US and Japan (OSHA and MRL). It was worth mentioning that one bangus sample contained OTC, OXA and Methoxychlor, but these were below the set limits by both Japan & US.. The rest of the samples were negative for the assayed chemicals.

Discussion The antibiotics OTC and OXA and the organochlorine pesticides were used in the aquaculture

production of popular species for culture based on the number of samples and fish species analyzed in this study. In Luzon (31 samples), the use of antibiotics seemed not as common compared to the use of pesticide. In Visayas (27 samples), OXA was commonly used compared to OTC. The popular use of OXA is possibly due to the perceived effectiveness compared to OTC which has been used for a long time wherein microorganisms developed resistance on its use. In samples from this region, all detected positive values (OTC, OXA and OCPs) were below the set values for MRLs. In samples from Mindanao (11), only one sample has OTC level above the set limits of OSHA and MRL. However, it should be noted that number of samples from Mindanao is less than half in number compared with that from Luzon or the Visayas region.

Among the antibiotics, OXA seemed preferred by farm operators to use compared to OTC. The antibiotic OXA has no established PEL but has an MRL so the use has a basis for regulation. For the OCPs, the most commonly used are Methoxychlor and groups of Endosulfan and Endrin.

Conclusion and Recommendations

This research work was conducted for only 3 years instead of 5 years and samples reported here are all from the regions of the Philippines islands. This work showed that the antibiotic OXA was commonly used compared with OTC with one sample exceeded the PEL and MRL limits (P. vannamei). On the other hand, the most common OCP detected in aquaculture products is Methoxychlor which was detected in samples from the three regions. For the other OCPs, namely Endrin ketone and Endosulfan I, only sample which was macrobrachium from Luzon was detected in harmful levels based on PEL and MRL.

Due to a decrease in budget allocation for this work activities to detect other chemicals or contaminants in feeds and aquaculture products were not conducted nor survey of the same from other less developed countries. It is recommended however, that for similar studies in the future heavy metals be determined in feeds and aquaculture by-products.

Acknowledgment The workers would like to acknowledge GOJ under the Japanese Trust Fund V and SEAFDEC/AQD for the support of conducting this study.

13

References Arthur, J. R., Lavilla-Pitogo, C. R. and Subasinghe, R. P. 2000. Use of Chemicals in Aquaculture in

Asia. Proceedings of the meeting on the Use of Chemicals in Aquaculture in Asia, 20-22 May 1996, Tigbauan, Iloilo, Philippines, aquaculture Department, SEAFDEC/AQD. 235 pp.

FAO. 1995. Code of Conduct on responsible Fisheries, FAO, Rome. 41 p. Carignan, G., Carrier, K and Sved, S. 1993. Assay of Oxytetracycline residues in salmon muscle by

Liquid Chromatography with Ultaviolet detection. J. of AOAC International. 76 (2) 325-328. Coloso. R. and Borlongan, I. 1999. Significant organotin contamination of sediment and tissues of

milkfish in brackish water ponds. Bull. Environ. Contam. Toxicol. 63:297-304. Londershausen, M. 2009. Presentation Title “Aquaculture in the light of regulatory requirements in

import countries” presented during a conference on “Innovations to Sustain Sea-food Supply” on March 12, 2009, BITEC, Bangkok.

Lacierda, E., de La Pena L. & Lumanlan-Mayo, S. 1996. Use of Chemicals in Aquaculture in the Philippines. In: Proceedings of the Meeting on the Use of Chemicals in Aquaculture in Asia (JR Arthur, CP Lavilla-Pitogo, CR Subasinghe, Eds.), 20-22 May 1996, Tigbauan, Iloilo, Philippines, Aquaculture Department, Southeast Asian Fisheries Development Center, Philippines. 155-184 p.

MFRD Report. 2008. Japanese Trust Fund II. Project on Research and Analysis of Chemical Residues and Contamination in Fish and Fish Products (2004-2008), Technical Compilation of Heavy Metals, Pesticide Residues, Histamine and Drug Residues in Fish and Fish Products in Southeast Asia. p 212.

Training Manual on Pesticide Residue Analysis, 6-15 June 2005, Singapore. MFRD. Singapore (Modified).

14

Tables and Figures

Table 1. Levels of Oxytetracycline (OTC), Oxolinic Acid (OXA), and the Organochlorine Pesticides (OCPs) in fish samples from Luzon

Table 2. Levels of Oxytetracycline (OTC), Oxolinic Acid (OXA), and the Organochlorine Pesticides

(OCPs) in fish samples from Visayas

15

Table 3. Levels of Oxytetracycline (OTC), Oxolinic Acid (OXA), and the Organochlorine Pesticides

(OCPs) in fish samples from Mindanao

Notes for tables 1,2 and 3: ppm = µg residue/g sample; No spls = Sample was not enough for the analysis; Red Values = samples exceed there MRLS for Agricultural chemicals (based on Japan); Black Values = samples DO NOT exceed there MRLS for Agricultural chemicals (based on Japan); “-“ = symbolizes absence of chemical contaminants in the samples (negative)

Table 4. The list of Minimum Residue Limits (MRLs) for Agricultural Chemicals (Japan Food Chemical Research) *

* Notes : Salmoniformes (such as salmon and trouts); Anguilliformes (such as eel); Perciformes (such as bonito, horse mackerel, mackerel, sea bass, sea bream and tuna); TWA= 8-hour time weighted average; PEL= Permissible Exposure Limits; TLV= Threshold Limit Values OSHA= Occupational Safety and Health Administration.

16

Fig. 1. Areas sampled in the Philippines showing results of OTC (Oxytetratcycline) analysis.

Fig. 2. Areas sampled in the Philippines showing results of OXA (Oxolinic acid) analysis.

Fig. 3. Areas sampled in the Philippines showing results of OCP analysis.

self-evaluation of the project … · toward establishment of micro-organism method ... study is...

Documents