Dela Cruz et al.

/ The Philippine Journal of Fisheries 29(1): in press January - June 2022

DOI: 10.31398/tpjf/29.1.2021-0009

RESEARCH ARTICLE

Method Standardization and Guidance Value Determination for

Regulation on Formaldehyde in Round Scad (Decapterus spp.)

James Dela Cruz *, Paul Bryan Paulo R. Cudia , Aira Mae M. Seguerra, Marc Lawrence J. Romero

Bureau of Fisheries and Natural Resources, National Fisheries Laboratory Division-Aquatic Toxicology Laboratory
860 Arcadia Bldg, Quezon Avenue, Paligsahan, Quezon City, Philippines, 1103

ABSTRACT
There are reports of traded Galunggong (Fil.), Decapterus spp., adulterated with formaldehyde (FA)
to lengthen shelf life. FA is a noxious substance with negative repercussions to general consumer health. This
warrants the determination of a guidance value due to a lack of a local regulatory value specific to Galunggong
amidst rising Filipino public concern in the wake of importations complicated by the natural occurrence of FA
in the marine fish post-mortem. Comparisons were made on FA levels of fish treated with different conditions
exposure and with colorimetric measurements using 405 nm and 412 nm via Nash’s method optimized for a
fish matrix with no significant difference in measurement to at most 30 ppm FA in an aqueous matrix. There
is also no significant difference in FA content of samples measured immediately from those frozen overnight.
Therefore, in line with the regulatory mandates of the Bureau of Fisheries and Aquatic Resources (DA-
BFAR) of the Department of Agriculture (Republic of the Philippines), a guidance value of 46.24 µg FA/g of
Galunggong or 46.24 ppm is suggested, which is obtained from FA measurements of fish subjected to minimal
anthropogenic treatments in different conditions of adulteration. Samples from different Metro Manila wet
markets serve as the baseline. The guidance value is suggested to be used as a reference for regulatory purposes
and can be improved with a more widespread sampling of fish from landing to selling.

*Corresponding Author: [email protected] Keywords: Galunggong, Round scads,

Received: April 5, 2021 Formaldehyde, Nash’s
Accepted: March 14, 2022 method, Colorimetric

1. INTRODUCTION 2020) and Southeast Asia (Tin 2019) being adulterated

T
with FA (Uddin et al. 2014; Sanyal et al. 2017) mainly
he Philippines is an archipelago rich with by dipping in various concentrations of formaldehyde
aquatic resources, with its coastal communities solution.
relying heavily on fish for protein. Logistics FA naturally occurs upon organic matter
to inland markets require preservation like cooling. combustion (Kaden et al. 2010; Salthammer et
Decapterus spp., known locally and officially as al. 2010). It is a popular preservative to biological
“Round Scad” or “Galunggong,” is a pelagic fish specimens (Fox et al. 1985), thus the association to
popular for being low-cost and highly available hence embalming. FA as a noxious substance (USNRCCT
monikered as the masses’ food with the colloquial 1980) affects mammals with irritation of the mucous
name “GG.” From the last decade, up until 2016 and membrane upon chronic exposure or lesions in severe
2018, there was a decrease in GG capture, hinting cases (Kerns et al. 1983), mild skin irritation (Maibach
at declining populations (PSA 2011; PSA 2019). 1983), and nervous system damage (Songur et al.
Since 2015, fishing seasons were imposed, dwindling 2010). FA is known in epidemiological studies (Ulm
local supply and subsequently increasing GG prices. 1989) upon great exposure (Kaden et al. 2010), a
Importations were made to curve this but, in turn, potential human carcinogen affecting the respiratory
made formaldehyde (FA) adulteration a public (Blair et al. 1990), gastrointestinal (Soffritti et al.
concern arising from reports of fish being traded in 1989), and bone marrow (Zhang et al. 2010) cells.
markets in South (Wahed et al. 2016; Times of India

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Dela Cruz et al. / The Philippine Journal of Fisheries 29(1): in press

FA is used in fish to preserve its apparent the standard, albeit more expensive. The study also
freshness (Bhowmik et al. 2017), sparring the product aims to determine a guidance value for regulatory
from lower prices. FA presence is not an immediate purposes.
indicator of adulteration as it occurs naturally in
fruits, meats, and fish (Chung and Chan 2009; Jaman 2 . M AT E R IA L S A N D M E T H O D S
et al. 2015; Wahed et al. 2016) and for marine fish,
like GG, is commonly formed at various levels from The study is comparative in nature. Different
osmoregulatory salts reducing in fish cells post samples from different sources like markets and landing
mortem (Castell et al. 1970; Sotelo et al. 1995; Benjakul sites were analyzed as a baseline. The FA content of
et al. 2004; Chung and Chan 2009: Cheng et al. 2015) fish treated with varying conditions of adulteration
as described in Figure 1. This reaction is catalyzed by was compared to the untreated controls. The matrix
cold storage (Leelapongwattana et al. 2005), further effect was also scrutinized. The FA derivative diacetyl-
degrading meat quality (Yasui and Lim 1987; Sotelo dihydrolutidine (DDH) (Nash 1952) maximally
et al. 1995; Summers et al. 2017). Some species have absorbs at λ=412 nm but was measured at λ=405
already elevated FA levels like Harpodon nehereus, nm, so comparisons were made in the measurements
or Bombay duck (Eng.), among other members between the two wavelengths.
of the genus Saurida (Lizardfishes), under family
Synodontidae (Chung and Chan 2009) and Gadidae 2.1 Chemicals and reagents
(Amano and Yamada 1964).
The concern is the anthropogenic adulteration Deionized distilled water (DDW), used
of FA in fish. The ambiguity of preparation and FA in the subsequent preparations and dilutions, was
ingredient grade are causes of concern. Although there obtained from a Milli-Q water system (Millipore, MA,
are regulatory limits placed for FA in fish in general in USA). The FA standards and simulating solutions
other countries, these values are on an ad hoc basis diluted in DDW were prepared from an AR grade
like in Sri Lanka, which has in place a value of 5.0 mg/ 37% w/w (3.7 x 105 ppm) solution of formaldehyde
kg (ppm) (Democratic Socialist Republic of Sri Lanka (stabilized in 10% methanol) purchased from Scharlau
2010) and in India with 4.0 mg/kg for freshwater fish (Barcelona, Spain). For the sample extractions, 5%
and 100.0 mg/kg (100.0 ppm) for marine fish (FSSAI w/w Trichloroacetic acid (TCA) solution was prepared
2019). The Philippines has yet to develop its regulatory from dissolved moist crystals of AR grade TCA
value. The analysis is done by Nash’s method (Castell purchased from Merck KGaA (Darmstadt, Germany)
and Smith 1973) via the Hantzsch pyridine synthesis in DDW. Nash’s reagent was prepared in DDW with
of a photoactive chromophore (Compton and Purdy the following final concentrations of 0.003% v/v of
1980). A.C.S. reagent grade glacial acetic acid purchased
Considering its regulatory mandate and from J.T. Baker (Phillipsburg, NJ, USA), 15.00% w/v
actions, DA-BFAR requires a relatively affordable of AR grade Ammonium acetate hygroscopic crystals
method, has a short turn-around time, and high sample purchased from Himedia (Kennett Square, PA, USA),
capacity. Therefore, this study aims to determine and 0.002% v/v of AR grade 99.5% Acetyl acetone
the differences in using various wavelengths in the solvent purchase from Loba Chemie PVT (Mumbai,
colorimetric analysis of FA in fish and the difference India).
between using a relatively less expensive method and

Figure 1. The degradation of Trimethylamine oxide to form volatile bases and Formaldehyde (Chung and Chan 2009)

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 Method Standardization and Guidance Value Determination for
Regulation on Formaldehyde in Round Scad (Decapterus spp.)

2.2 Instruments qualified batch of multiple fish in a bag until five (n=5)
fish were obtained. The fish were then fileted on both
Instruments and wares used are the sides. The resulting two filets were combined, minced,
following: BioHomogenizer®, hand-held homogenizer and mixed on a chopping board with a kitchen knife
manufactured by ESGE Switzerland, Lab. Centrifuge® until the sample was pureed into a clay-like solid of
by Digisystem Laboratory Instrument Inc. BioTek homogeneous consistency. The resulting puree was
Instruments Inc. (Winooski, VT, USA). Plate reader coned and quartered until 5 g was weighed into a
with model no. ELx800 carried out measurements 50 mL centrifuge tube. The sample was added with
using the 96-well plate format designated in this study 20 mL of TCA and homogenized with a hand-held
as “PR” and the Shimadzu Asia-Pacific (Singapore) homogenizer at the highest setting for 15 seconds. The
model no. UV-1800 Spectrophotometer carried out resulting homogenate was left to stand for 30 minutes
measurements using a quartz cuvette designated as at room temperature. The homogenate was then
“SPM.” placed at a centrifuge set at 3000 RPM for 10 minutes.
The resulting supernatant was decanted and filtered
2.3 Study area and baseline data with a Whatman No. 41 filter paper into a 100 mL
volumetric flask and diluted to mark with deionized
On August 24, 2018, in conjunction with the distilled water. The precipitate pellet was disposed of.
monitoring order of DA-BFAR, samples of GG were A 3 mL aliquot was obtained from the diluted sample
obtained from Farmer’s Market (14.62° N, 121.05° solution into a capped test tube. Three (3) mL of Nash’s
E) and Balintawak market (14.66° N, 121.00° E) in reagent was added. The resulting solution was then
Quezon City and Navotas Fish Port (14.64° N, 120.95° heated for 15 minutes at 60° C. The sample was then
E) in Navotas City. Subsequently, Muñoz Market cooled and set for analysis. For the analysis using PR,
(14.65° N, 121.01° E) in Quezon City was sampled the 96-well plate was added with the standards and
monthly for 2019. A total of four sampling sites were samples. Each sample is loaded into the plate at five
identified, with three sites sampled once and one site trials (t=5), with one well per trial, and analyzed at
sampled monthly. The sites were selected considering 405 nm. For the analysis using an SPM, measurements
popularity, proximity to areas of high population were done with a quartz cuvette and with the 405 nm
hence high patronage, large output, and proximity and 412 nm beam at four trials (t=4) each.
from the laboratory for practical reasons. Quezon
City and Navotas City are part of the populous Metro 2.5 Standards preparation and analysis
Manila. Navotas Fish port is the largest and primary
fish landing site of Metro Manila. The state of the A 1000 ppm FA stock solution is prepared by
GG in the stalls was displayed in the open air but transferring 0.270 mL of 37% AR grade FA into a 100
surrounded by tubed ice. To the best of the sampler’s mL volumetric flask and diluted to mark. The stock
inquiry, all the GG samples were landed the day prior, solution is used to prepare 0 ppm, 3 ppm, 6 ppm, 12
transported under ice, and displayed on the morning of ppm, 24 ppm, and 30 ppm standard solutions. Three
the sampling day. From the selected stalls that display (3) mL aliquots were obtained from the standard
fresh GG with tubed ice, the stalls to be sampled were solutions and were added with 3 mL Nash’s reagent
selected based on price and ease of access. All stalls with the resulting solution heated like the treatment
sampled obtained their fish from Navotas fish port. on the samples and subsequently measured alongside
Most GG landed in the harbor are caught from the the samples.
West to the southwest seaboard of Luzon, Philippines.
The sampling of the stalls is done to obtain a baseline 2.6 Matrix effect and recovery tests
value and evaluate the applicability of the suggested
guidance value. The samples obtained here were only The matrix effect is the “effect of the matrix”
analyzed at 405 nm using the plate reader. on the analysis of the analyte, which may cause
further positive or negative deviations from the true
2.4 Sample preparation and analysis value. This effect is evaluated by measurements of
background and spiked samples at different dilutions
A fresh fish batch from a selected stall was (dilution factor (DF) of 1, 5, 10, and 20), and in this
set and evaluated by weighing the fish to know if each study was done with PR at 405 nm. The obtained
fish in the batch could provide a representative 5 g measurements are then compared to each other for
of fish flesh. The fish were grabbed blindly from the any significant differences, the presence of which is

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indicative of the matrix effect, the evaluation of which analyzed with the method. Note that the FA solutions
is critical in determining the possible interference that were prepared with the assumption that the AR grade
must be taken into account during measurement and solution of FA available in the market with an aqueous
calculations. concentration of 37% is 100% for convenience. The
Method validation for this study is somewhat latter two conditions were also aimed to simulate
limited since certified reference materials for FA minimal conditions of FA adulteration. This is done
in fish matrix specifically for GG meat are not yet to simulate and obtain the likely FA levels of GG
available. CRMs for FA exist but for wastewater or subjected to the minimum of FA adulteration.
effluent matrices and may not necessarily apply to
the method of this study like provided by Supelco® 2.9 Statistics
“Formaldehyde in Water” CRM, which is more
applicable to chromatographic methods. Instead, on Data management is done in Microsoft
account of costs, availability, and time, the study opted Excel 2021. Statistical analyses and comparisons of
to use the earlier mentioned FA AR grade reagent with the differences of the conditions and matrix effect
a known concentration for spiking. Aqueous solutions were carried out using T-test at α=0.05 and One-way
of FA at 15 ppm were prepared and measured in PR at ANOVA at α=0.05 if the comparison is among groups
405 nm and SPM at 405 nm and 412 nm. This is done (sample size n=5) with GraphPad Prism 8.0.2 (Free
to gauge the applicability of using 405 nm from the Trial version). The software automatically tests for
standard 412 nm probing wavelength and gauge any homoscedasticity upon statistical comparisons. In
differences between using PR versus SPM. cases where the variance is equal, comparisons were
made with the variance of the mean; otherwise, it
2.7 Comparison of using λ=405 nm vs resorts to the median, as is the case for large variations.
λ=412 nm and SPM vs. PR Calibration was done by plotting the measured
absorbance of the standards. The resulting plot was
A reagent blank was prepared, denoted with then linearly regressed using both MS Excel and
"RB" sample code. Two samples of GG represented Prism to obtain linear parameters used to calculate the
as "AG1" and "AG2" were treated with FA by soaking samples' concentrations.
them in a 5% solution for an hour with another
separate sample acting as control denoted "G." Two 3 . R E S U LT S
sets of each of the codes were prepared at three
trials to be analyzed in the following conditions: the 3.1 Baseline data of formaldehyde in GG
first set was analyzed at PR 405 nm and the second
set was analyzed in an SPM at 405 nm and 412 nm, Results for the sample analysis from the three
respectively. The different linearity parameters of the markets and the landing site that would serve as the
calibration curves were also determined by measuring baseline data were summarized in Figure 2. Measured
the standards at PR 405 nm, SPM 405 nm, and SPM FA content in the samples in the 2019 series in Figure
412 nm. 2 displayed no significant trend. It was generally
consistent throughout the year, suggesting no effect
2.8 Determination of a recommended of season changes and time of capture, sale, and
guidance value sampling.

Samples of GG were selected from a batch 3.2 Matrix effects and standards spiking
purchased in Muñoz Market. First, a fresh fish batch
from a selected stall was set and evaluated by weighing Figure 3 summarizes the data obtained from
the fish to know if each fish in the batch could provide investigating the “matrix effect” of the GG matrix. The
a representative 5 g of fish flesh. Then, the fish were p-values obtained showed insignificant differences of
grabbed blindly from the qualified batch of multiple measurement among the dilutions, which suggests
fish in a bag until five (n=5) fish were obtained. Next, insignificant effects of the matrix at the method’s
the sampled fish were directly soaked in a solution analytical concentrations. This supports the use of the
of FA at different concentrations and other different aqueous solution of FA without the matrix in standards
conditions like exposure time and subsequent storage spiking. As for the measured FA concentrations of
and freezing specified in table 1 to simulate different the theoretically 15 ppm aqueous FA solutions, there
conditions of adulteration. Finally, the fish were was no significant difference with the measurements

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 Method Standardization and Guidance Value Determination for
Regulation on Formaldehyde in Round Scad (Decapterus spp.)

carried out using PR at 405 nm and SPM at 405 412 nm was insignificant (p=0.2774) (see Figure 4).
nm and 412 nm with a p-value of 0.2202 upon As for accuracy, with the prepared standard having a
comparison with One-way ANOVA, see figure 4. As theoretical concentration of 15 ppm, it can be seen in
far as repeatability is concerned, the resulting repeated Figure 4 that the mean with the standard deviation of
measurements (n=10) of 15 ppm FA solutions at PR the obtained values encompassed the theoretical value
405 nm resulted in insignificant differences with one for PR 405 nm, SPM 405 nm, and SPM 412 nm. The
another (p=0.1425) (see Figure 4). As for robustness, limit of detection is calculated for PR 405 nm, SPM
the difference in the measurements of the 15 ppm 405 nm, and SPM 412 nm is 0.56 ± 0.17 μg/mL, 0.71
solutions using PR 405 nm, SPM 405 nm, and SPM ± 0.21 μg/mL and 0.47 ± 0.14 μg/mL, respectively. The

Figure 2. Summary of the measurements of the market monitoring and landing samples which serve as the baseline measured using a
Plate reader at 405 nm (PR 405 nm). Seen also is a red line marking the obtained guidance value which is significantly above the obtained
background levels of Formaldehyde for reference. Note: n is the sample size, t is the number of measurement trials per sample.

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Figure 3. Fomaldehyde levels in μg/g or ppm of samples (from fish subjected to different adulteration conditions) at different dilutions post
extraction with p-value from using One-way ANOVA at α=0.05. Note: DF means Dilution Factor. The red line indicates the guidance value
obtained. Data obtained is used to evaluate the matrix effect.

limit of quantification is calculated to be 1.68 ± 0.17 differences in the obtained measurements of the
μg/mL, 2.15 ± 0.21 μg/mL and 1.42 ± 0.14 μg/mL, standards between the two different wavelengths. The
respectively. difference for PR 405 against the two SPM was no
longer analyzed as the difference is noticeable.
3.3 Comparisons of the different wavelengths
and instruments 3.4 Determination of a guidance value

Figure 5 summarizes the measured values After analysis of the fish samples subjected
of the different FA adulteration simulations as to the conditions of FA exposure simulations,
mentioned in section 2.8. Figure 5 also summarizes conditions 1S and 5S as specified in Table 1 resulted
the comparisons of the various conditions under in FA levels that were significantly larger than that of
different measurement parameters like wavelength the control, using t=test at α=0.05 with p<0.001 for
and instrument. Figure 6 summarizes the difference of both conditions. Further, the sample introduced to
the generated calibration curves in PR 405 nm, SPM a higher concentration of FA had higher levels (see
405 nm, and SPM 412 nm. Said calibration curves Figure 7). Apparent FA levels in conditions 1Da and
are virtually linear as far as the Pearson’s linearity 1Db are also significantly greater than that of the
coefficient is concerned, suggesting the feasibility of controls, using t=test at α=0.05, p-values of 0.007 and
calibration for PR and compliance to the Beer-Lambert 0.009, respectively, against the control. There is no
relation. As far as differences are concerned, for SPM significant difference between treatments 1Da and
405 nm and SPM 412 nm, there were no significant

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 Method Standardization and Guidance Value Determination for
Regulation on Formaldehyde in Round Scad (Decapterus spp.)

Figure 4. Box graph showing the recoveries of the measurements of 15 ppm Formaldehyde at different
measurement conditions. Note: n is the sample size, t is the number of measurement trials per sample. Shown
in the parenthesis is the p-value of the comparison of the means between using Plate reader at 405 nm (PR
405 nm) and using the Spectrophotometer at 405 nm and at 412 nm (SPM 405 nm and SPM 412 nm). Shown
in square bracket is the p-value of the comparisons of the means obtained for the Plate reader at 405 nm.

Figure 5. Summary of the Formaldehyde content measured at the two different wavelengths and instruments.
Comparinsons of the mean (median* in cases where the mean fails tests for homoscedasticity) of measurement
conditions and samples between a. Spectrophotometer at 405 nm (SPM 405 nm) vs. the same instrument at
412 nm (SPM 412 nm), Plate reader at 405 nm (PR 405 nm) vs Spectrophotometer at b. 405 nm (SPM 405 nm)
and at c. 412 nm (SPM 412 nm) result in the above p-values. Italicized values indicate significant differences
between the two conditions. Note: n is the sample size, t is the number of measurement trials per sample

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Figure 6. Calibration curves of the measurements using the two different wavelengths and instruments. In parenthesis are the concentrations
of the standards and p-values of the comparison between the absorbances measured between 405 nm and 412 nm using the the
Spectrophotometer (SPM 405 nm and SPM 412 nm). The calibration curve obtained from measurements using the Plate reader at 405 nm
(PR 405 nm) is no longer compared due to the obvious difference. Note: n is the sample size, t is the number of measurement trials per
sample.

Table 1. Different conditions and parameters simulating FA adulteration in Galunggong (*<1 sec: rapid immersion of the fish being held from
the tail, dunked quickly headfirst until fully submerged and immediately removed with excess solution removed by shaking)

Condition FA (%v/v concentration) Exposure time (sec or hr)

Control Control Control (0 sec)
1S 1% 1 hr
5S 5% 1 hr
1Da 1% Dipped (<1 sec*)
1Db 1% Dipped (<1 sec*) + overnight freezing

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 Method Standardization and Guidance Value Determination for
Regulation on Formaldehyde in Round Scad (Decapterus spp.)

Figure 7. Amount of Formaldehyde in the samples analyzed that were subjected to the simulated
conditions of adulteration. Note that the data presented only accounts for the undiluted sample with a
dilution factor of 1.

1Db (p>0.05). The lower limit value obtained from the Another method proposed is comparing the amount
minimum of FA adulteration that was not subjected to of Dimethylamine (DMA) on account of the naturally
refrigeration is 46.24 µg FA/g or 46.24 ppm (Note: 1 stoichiometrically equal amounts of the two cleavage
µg/g is equivalent to 1 ppm). products of Trimethylamine oxide (TMAO) even in
different conditions. Cases where the amount of FA
4. DISCUSSION in the fish is higher than DMA, indicate adulteration
(Shen et al. 2015) with measurements carried out
A guidance value is needed on account with HPLC (Chung and Chan 2009). However,
of the requirements of the regulatory functions concerns in the determination of FA are accessibility
of DA-BFAR and considering the biochemical and affordability, especially for DA-BFAR’s central
nuances of FA detection. Methods like Isotope Ratio and regional laboratories on the scale of national
Mass Spectrometry (IRMS) can be used to detect regulation and the sheer number of samples requiring
anthropogenic FA in fish as well as a proposed short turnaround times. A more inexpensive and faster
superiority of using High Performance Liquid alternative suitable for the DA-BFAR’s purposes would
Chromatography (HPLC) (Hoque et al. 2018). be to use spectrophotometric/colorimetric methods to

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determine anthropogenic FA chemically to a degree substances worthy of studying compared to food grade
rather than chromatographic or nuclear analytical FA if ever it exists. The probability of unprofessional
methods. Going further in this vein is the slight handling and preparation of the reagent and illegal
preference for PR over SPM in terms of availability, adulteration leading to surpassing the threshold
cost, and turnaround time. PR has high sample reference dose anyway is not farfetched.
capacity due to the 96-well plate format, though SPMs The proposed guidance value is only
can also read in 96-well plate format instead of cuvette recommended for Galunggong as this is the fish in the
format, albeit it tends to be more expensive. study. Due to the considerations of DA-BFAR to use
The measured levels of FA in the fish that was a more available wavelength via relatively less costly
adulterated in the laboratory would be significantly PR for the benefit of DA-BFAR’s regional laboratories
elevated statistically compared to the baseline, and field activities, 405 nm is the available option for
whose measurement can be attributed solely to the measuring wavelength when conducting colorimetric
background and naturally produced postmortem analysis as opposed to the method’s established 412
levels of FA in the flesh as well as a small amount nm. The measured absorbance of the standards at
of matrix interference. A guidance value of 46.24 SPM 405 nm against SPM 412 nm are insignificantly
µg of FA/g fish is obtained from the minimal of the different though it can be observed that the variance
measurements from non-refrigerated adulterated increases at higher concentrations. This entails further
samples, which is value due to the incorporation of study on higher concentrations, but for the time
the natural background FA levels with the presumed being, the insignificant difference is presumed from
levels from minimal conditions of adulteration; hence minimum to at most 30 ppm. Additionally, comparing
any measurement above the mentioned value can and the slopes of the calibration curves generated from
may be grounds for possible adulteration of GG. The measurements using PR and SPM would be difficult
US-EPA’s reference dose translates to 0.2 mg per kg considering the rather large difference between the
body weight per day, and the average body weight of two instruments (see Figure 6). According to the Beer-
Filipino males and females as per the Department of Lambert relationship, this could be explained by the
Science and Technology-Food and Nutrition Research fact that path lengths are a factor in the relationship
Institute’s (DOST-FNRI) 8th National Nutrition between absorbance and concentration. In short, the
Survey is at estimated values of 61.3 kg and 54.3 kg, path length is related to the slope, which is different
respectively (FNRI-DOST 2013). Considering the between PR and SPM, supported by the fact that PR
data and assuming an ordinary Filipino consumes 100 uses a 96-well plate while SPM utilizes a cuvette. The
g of GG for a day, they would have consumed about material of the 96-well plate and the cuvette are also
0.0745 mg and 0.0852 mg of FA per kg body weight for factors. However, differences caused by these factors
Filipino males and females, respectively. This suggests are compensated with calibration. The same cannot
that amidst being a value obtained from the sample be said about varying the wavelength as this factor
subjected to presumably the minimum conditions of changes the molar extinction coefficient in the Beer-
adulteration, consumption is still deemed safe as far Lambert relation. However, concerning 405 nm and
as the reference dose is concerned. In the same vein, 412 nm, the difference is not significantly different at
this would imply that to reach the threshold that is the the range of concentration used in this study.
reference dose of 0.2 mg per kg body weight per day, Due to the failure of some means to
one who is 50 kg has to consume for a day 100 g of homoscedastic tests, the medians are used instead with
fish that has an FA content of 100 µg/g fish in which, merit because results varied widely among samples
at least with the presumed parameters, is parallel with (see Figure 5). Representativeness of the sample was
the FA oral exposure limit of the European Food Safety primarily limited to the individual fish themselves;
Authority (EFSA) of 100 mg per day (EFSA 2014). this was the rationale of filleting the fish completely,
The guidance value is below the reference dosage rationalized further by the fact that this is generally
threshold of the US-EPA. However, the concern is the the only part of the edible fish, then homogenizing it
act of adulteration and not immediately the risk of before the sample was obtained. It is recommended to
surpassing the threshold since merely adulterating the increase the sample size and expand the parameters
fish products is a violation under RA 10654 and RA on which the fish would be subjected.
10611. In addition, the risk posed by using FA in fish It can be recommended to widen the
is aligned to the risks posed by using AR and lower sampling reach and incorporate other fish dealership
grades of FA, which may have impurities that are also facilities like fish ports and ambulatory hawkers. Other

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 Method Standardization and Guidance Value Determination for
Regulation on Formaldehyde in Round Scad (Decapterus spp.)

methods of FA introduction to fish, such as injection, AU T H O R C O N T R I BU T I O N S

are also recommended to be studied. Additional
comparisons and testing for the use of 405 nm in PR Dela Cruz J: Conceptualization, Methodology,
against 412 in SPM are also recommended. It is also Software, Writing—reviewing, Writing—Original
recommended to conduct a further and detailed risk Draft Creation, Editing, Formal Analysis. Cudia
assessment and profiling considering the Acceptable PBKR: Visualization, Investigation, Methodology.
Daily Intake (ADI) regarding the risks posed by FA Seguerra AMM: Visualization, Investigation, Data
adulteration in fish. Other factors like the morphology Curation. Romero MLJ: Supervision, Writing—
of the fish, other aspects of the fish’s skin, i.e., scale, reviewing, Editing, Project Administration.
keratinized layers, thickness, openings, etc., and other
physicochemical conditions may come into play that CONFLICT OF INTERESTS
would cause the variation of the FA levels of the fish
given the same conditions of exposure to FA; thus, The authors declare that there is no known
warranting the need of further testing to other species. conflict of interest.
The species that have naturally elevated levels of
FA, as well as other fishes and probable methods of E T H I C S S TAT E M E N T
adulteration, have yet to be tested if any other values
optimized for these circumstances can be obtained. No animal or human studies were carried out
Factors like pre-consumption treatment of the fish like by the authors. The study and the authors adhered with
drying, smoking, and down to cooking like boiling the utmost compliance to modern ethical standards.
in soup or frying would influence the amount of FA The test fishes are all procured post-mortem and are
in the fish, which would pose different levels of risk already available for human consumption. To the
warranting not only additional tests with these factors best of the authors’ knowledge, the authors release
in mind but also additional points for risk assessments themselves from any ethical concerns involving
the monger of the fish prior to purchase. On the
5. CONCLUSION environment: chemicals, reagents, and wastes are
stored and disposed of properly in compliance with
The guidance value of 46.24 μg/g obtained environmental regulations. No identifying information
from the study would be helpful and will provide of the fishmongers is recorded for and included in the
for the regulatory functions of DA-BFAR and other article; therefore, consent is not necessary; only the
regulatory entities. As far as what was covered in the general location of the sampling site is recorded for
study is concerned, there is a statistically significant reproducibility.
difference between presumably unadulterated fish
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