Analysis Of Ddts In Different Parts Of Mola Fish Samples

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Analysis Of Ddts In Different Parts Of Mola Fish Samples

1.1 Reagents, chemicals and solvents

All the chemicals, reagents and solvents used for analysis in the present work were(purity 99.99%) of either pesticide grade or HPLC or analytical grade. Ethyl acetate, n-hexane and acetone were purchased from Merck (Darmstadt, Germany). Sulfuric acid (98%, w/w) and silica sand were bought from BDH (Poole, UK) and Kanto Chemical Co. Int. (Tokyo, Japan), respectively. Anhydrous sodium sulphate was heated at 300 0C for at least 4 hours in an oven and cooled before use.

2.1 Glass apparatus

All required glass apparatus including graduated test tubes were cleaned with water using detergent, rinsed about six times with water, then twice with distilled water and finally with redistilled acetone. All glassware was heated at 300 ºC overnight, cooled and stored by covering with aluminum foil prior to use.

2.2 Instruments and equipment

For extraction of DDTs from all kind of samples a centrifuge machine (Hettich Universal 2S, Germany) was used. For clean-up, a small centrifuge machine, model 800, Xiangshui Fada Medical Apparatus Factory, China was used in the present work.

All glass apparatus were dried after cleaning in an oven (EYELA, Rikakikai Co. Ltd. NDO-450 ND, Tokyo Japan) at 105 °C. Anhydrous sodium sulphate was dried at 300 °C for 6 hours in a furnace (Carbolite, England). Silica-gel was also dried at 300 °C about an hour in the same furnace before impregnated with conc. sulphuric acid. Small amount of solvent was concentrated at 30 °C by the flow of nitrogen gas in a block heater (Grant Instrument, Cambridg) Ltd. England). All the samples were cleaned-up by inversion using an inverter (Boule Mixter 440, Boule Medical, Stockholm, Sweden). Mixing of small amount of solvents were done by a Vortex machine purchased from VWR International, Germany. Vegetable samples were extracted with Ultra Turrax, purchased from Sweden (IKA-WERK).

2.3 Evaporation

All the evaporations were carried out under reduced pressure using rotary vacuum evaporator at bath temperature not exceeding 40 0C. Small volume of organic solvent was removed by blowing nitrogen gas putting the graduated test tubes in block heater

(30 0C)

2.4 Analysis of Pesticide Residue in mola Fish Sample

2.5.1 Sample collection

Mola fish samples were purchased from Hatir Pool Baza (sample-1), Polashi Baza (sample-2), Kaptan Bazar (sample-3), Uttar (sample-4) of Dhaka and Joydebpur Baza r(sample-5), Shalna Bazar (sample-6), Tongi Bazar (sample-7) of Gazipur and kept in a covered jar and transferred immediately to the laboratory.

Table-2.1: Sample number, local and scientific name, date and area of collection of the collected fish sample.

Sample N0 Local name Scientific name Area of Collection Date

of Collection

1 Mola Anblypharyngodon mola Hatir pool bazar 21.04.10
2 Polashi 26.04.10
3 Kaptan bazar 05.05.10
4 Joydebpur 25.10.10
5 Uttara 27.10.10
6 Shalna 01.11.10
7 Tongi 07.11.10

2.5.2 Sample storage

All fish samples were wrapped with aluminium foil. All the collected fish samples were stored in a freezer at a temperature below -15 0C until dissection.

2.6.1 Extraction procedure of mola fish sample[39]

Each of the collected fish sample was taken out from the freezer. The head, body and gut were divided into several portions of 10 g for replicate analysis. Representative sample (10 g) was taken in a mortar.. Then 10 g silica sand and 30 g anhydrous sodium sulfate were added to the samples and the mixture was ground until the sample floated freely (additional sodium sulfate was added wherever needed). The powdery sample was taken in a conical flask (ground joint 500 mL) having a stopper and extracted successively with 60, 20, 0mL of ethyl acetate by shaking 3 minutes each time. The extracts were combined and filtered using filter paper with few gram of anhydrous sodium sulfate

2.6.2 Eveporation

The filtrate was concentrated to by evaporation and the amount of fat was measured.Then the extract was reconstituted in n-hexane before injection to GC-ECD.

Fig-2.1:The evaporation process was carried out in Rotavapor

The extraction procedure is presented in Scheme-1.

Scheme-1 Extraction procedure of fish samples

Homogenized by a kitchen blender

2.6.3 Clean-up procedure of fish samples[39]

The extract in n-hexane was cleaned up by concentrated sulphuric acid treatment to remove fat and other impurities present in the extract. The clean extract will contain only organo halogenated compound.

2.6.3.1 Saturation of sulphuric acid

Concentrated Sulphuric acid (80 mL, 98%) was taken in a reagent bottle and 20 mL of n-hexane was added to the acid. It was shaken about one minute and kept to separate the two phases. The lower sulphuric acid phase, saturated with n-hexane was collected and the upper phase was discarded.

2.6.3.2 Clean up

The extract (~1 mL; in a graduated test tube) was treated with concentrated sulphuric acid (1 mL) saturated with n-hexane and the test tube was inverted by an inverter carefully ~40 second (not shaked). Then the content was centrifuged for 5 minutes to separate the two layers. The upper clean organic phase was taken into a clean and dried vial.

2.6.3.3 Additional clean up

Additional clean up was carried out through silica gel: sulfuric acid (2:1 g/g) column. The extract was then concentrated to 1mL through air blow before injection in GC equipped with ECD.

The total clean-up procedure is demonstrated in Scheme-2.

Scheme-2 Clean-up procedure for fish sample extract

2.7 Determination and quantification of residual pesticide in mola fish samples using GC-ECD

A GC-17 a SHIMADZU Gas Chromatograph (Fig.2.2) having Electron Capture Detector (ECD) was used for identification and quantification of organo halogenated compound. ECD was very sensitive and Limit Of Detection (LOD) was 1 fg level. Capillary column of 30 meter length and 0.01/0.02µm Inner Diameter (ID) was used for the analysis.About 1µl sample was injected to the column.

2.7.1 GC Condition for 17 A

• Temperature of the detector: 3100C

• Injector temperature: 2800C

• Column oven- Initial temperature: 800C, hold 1 min, then 200C rise per min to 300 and hold 5 min.

• Column specification: DV 5

• Length of the column: 30 m

• Internal diameter of the column: 0.25 mm

• Column thickness: 1.5 µm

• Carrier gas: Nitrogen

• Flow rate: 2 ml/min

• Injection :Split mode

• Split ratio: 1:70

• Injection volume: 1 µl

Fig-2.2: Gas Chromatograph (Model GC-17A)

2.8 Pesticide Standards

Primary standards (99.999% purity) of the highest available purity were purchased from Dr. Erhenstorfer, Germany. The standards were stored in a freezer at 00C. Standard mixtures of various concentrations (0.5, 0.25, 0.125, 0.0625 and 0.0312 ppm) were prepared in n-hexane for calibration curve and identification of samples.

All the solutions were kept or preserved in leveled screw cap test tubes with proper marking by permanent ink and stored in a refrigerator. During storing of standard sample if there is any loss of solvent, adding more solvent up to the mark and made homogeneous by vortex.

2.8.1 Preparation of Standard Solutions

2.8.1.1 Preparation of stock standard solutions

The known amount of the standard was dissolved in a definite amount of solvent (cyclohexane or acetone) and the concentrations of the standards were calculated. These stock solutions were transferred to different narrow test tube, with PTFE lined screw cap. These solutions were labeled indicating name of the standard, solvent, concentration, date of preparation and signature. The meniscus were of the solutions were marked withpermanent ink. These solutions were stored in the freezer of a refrigerator.

2.8.1.2 Preparation of middle and working standard solutions

The stock solution was kept in the outside of the refrigerator to reach room temperature. Then a definite amount of the solutions was withdrawn after checking the meniscus mark and put a new mark after withdrawing. The withdrawn solution was dilute with the appropriate solvent for the analytical work. This solution was transferred to a narrow test tube, with a PTFE lined screw cap. This solution was labeled indicating substance, solvent, concentration, date of preparation and signature. The meniscus of the solution was marked with permanent ink.

Similarly, the working solutions were prepared from middle standard solutions; both middle and working standard solutions were kept in the residue laboratory.

2.9.1 Identification of DDTS by GC

For identification of DDTs present in the spiked samples, reference standard samples were used. By comparing the retention time of peak present in the sample chromatogram with the chromatogram of standard DDT mixture (DDD, DDE and DDT) were identified.

2.9.2 Quantification by GC

Standard solutions were injected before, intermittently and after a set of samples. The solvent compositions for the external standards and the extracts were kept same. Equal volume of the sample solution and the standard solutions were injected in all cases.

Quantitative determination was carried out by comparing the peak height of the blood samples with that of the peak height of the standard solution. By using sigma plot software, calibration curve of Standards were made. From the calibration curve, the amount of DDTs present in the sample extracts was also calculated.

Amount of DDTs were calculated by using the following formula

Amount of DDTs =

Where, Conc.Matrix =

2.9.3 Recovery experiments of fish samples

Known amount of pesticide standards were added drop by drop over to the fish control sample (10 g) and allowed the sample to stand for 30 min to let the pesticides be absorbed into the samples. The samples were extracted and cleaned-up by following the same procedure as described above. Reagent blank samples were also analyzed. The recovery of each of the pesticide was calculated by using the following formula.

Area Sample × Conc. Std. 100

Recovery = ×

Area Std. × Conc. Matrix Known amount of Std

The percentage of recovery was found 126, 93 and 66% for DDE, DDD and DDT.

2.9.4 Control sample

A blank experiment was done with local Snakehead fish sample. Its chromatogram showed that concentration of DDE, DDD and DDT present in the sample were below the detection limit

2.9.5 Standard analysis

To compare the retention times of DDE, DDD and DDT of the Mola fish samples, the standard solutions of DDE,DDD and DDT were prepared and injected into GC. The retention times of the standard solutions were recorded and tabulated in the following Table:2.2

Table-2.2: Retention times of standard solutions of DDE, DDD and DDT

Standards Retention Time(min)
DDE 11.27
DDD 11.73
DDT 12.16

The solutions of standard mixture of DDTs of various concentrations were prepared and injected into GC to find out the peak areas of DDE, DDD and DDT and to draw a calibration curve. The peak areas of the standard mixture of DDT of various concentrations were recorded and tabulated in Table:2.3

Table-2.3: Peak areas of DDE, DDD and DDT at different conc. for calibration curve.

Concentration

(ppm)

DDE

(Paek Area)

DDD

(Peak Area)

DDT

(Peak Area)

0.0312 3925 1834 1843
0.0626 6271 3189 3021
0.125 9567 5124 4829
0.25 14859 8748 8410
0.5 23978 15088 14326

2.9.6 Fat content

Table-2.4: Amount of fat in the body of different Mola fish sample

Sample ID Amount of fat

(mg/g f.w.)

Average fat (mg/g f.w)
B1H 11.00 12.50
B2H 14.00
B1P 55.00 54.50
B2P 54.00
B1K 24.00 27.00
B2K 30.00
B1J 25.00 25.50
B2J 26.00
B1U 13.00 12.50
B2U 12.00
B1S 11.00 12.00
B2S 13.00
B1T 27.00 25.50
B2T 24.00

f.w-fresh weight

Table-2.5: Amount of fat in the head of different Mola fish sample

Sample ID Amount of fat

(mg/g f.w)

Average fat (mg/g f.w)
H1H 78.00 77.50
H2H 77.00
H1P 44.00 43.00
H2P 42.00
H1K 76.00 75.00
H2K 74.00
H1J 77.00 78.00
H2J 79.00
H1U 69.00 67.50
H2U 66.00
H1S 70.00 72.00
H2S 74.00
H1T 0.0610 0.0625
H2T 0.0640

f.w-fresh weight

Table-2.6: Amount of fat in the gut of different Mola fish sample

Sample ID Amount of fat

(mg/g f.w)

Average fat (mg/g f.w)
G1H 103.00 102.00
G2H 101.00
G1P 51.00 50.00
G2P 49.00
G1K 133.00 127.50
G2K 122.00
G1J 262.00 262.50
G2J 263.00
G1U 259.00 260.00
G2U 261.00
G1S 174.00 172.50
G2S 171.00
G1T 295.00 293.50
G2T 292.00

f.w-fresh weight

2.9.7 Calibration Curve

Five different concentrations (0.03125, 0.0625, 0.125, 0.25, and0.5 ppm) of DDE, DDD and DDT were used to prepare calibration curve for them respectively. The calibration curve of each of standard was made by plotting area vs concentration. By using the formula y = mx + c the concentration is calculated.

Here, y = Peak area

x = Concentration

m = Slope of the calibration curve

c = Intercept

Fig-2.3: Calibration curve for standard DDT.

b[0] 1376.4423379071

b[1] 26372.2394038036

r ² 0.9960456261

curve in Fig:2.3 shows linear regration.

Fig-2.4: Calibration curve for standard DDE.

b[0] 3353.1663453217

b[1] 44608.4115550652

r ² 0.9954840999

curve in Fig:2.4 shows linear regration.

Fig-2.4: Calibration curve for standard DDD

b[0] 1405.2587784694

b[1] 27827.7135415021

r ² 0.9959721618

curve in Fig:2.5 shows linear regration.

2.9.7 Limit of detection (LOD)

To find the limit of detection, gradually diluted mixture of DDTs were injected to the GC and peak the area of each solution was considered acceptable if it was three times higher than the noise level. Analyzing the peak areas of all the solutions the detection limit was found to be 0.39 ng for DDE & DDD and for DDT it was 1.56 ng.

2.9.8 Limit of quantification (LOQ)

To find the limit of quantification, gradually diluted standard mixture of DDTs were injected into GC and the peak areas of each solution were considered acceptable if it was ten times higher than the noise level and 3 times of LOD. Analyzing the peak areas, the quantification limit was found 1.36 and 4.89 ppb for DDE, DDD and DDT respectively.

3.1 General

Because of the enormous increase in the world population, the demand for food production has increased. With the increase of population, traditional way of cultivation has been changed and large amount of fertilizers and pesticides are being used to increase food production. But indiscriminate use of various kinds of hazardous pesticides is threatening to the environment.[40]Bangladesh is mainly an agricultural country. Farmers used DDT to protect crops against pests. However, there are some reports in the daily newspaper that DDT still might be used illegally.

The major exposure of the persistent organohalogen compounds to humans is via contaminated food, drinking water, inhalation and dermal uptake. Aquatic ecosystems are the reservoirs of many contaminants. DDTs enter into aquatic ecosystems either due to direct discharge or hydrologic and atmospheric processes. Fish bioaccumulate many pollutants, especially DDTs and other organochlorine substances, thus monitoring of fish for the presence of organochlorine compounds can give the level of exposure of these pollutants in the environment. Therefore, the present study was aimed to the assess the level of DDTs in different parts of mola fish.

3.2 Sample collection and extraction

A total of seven mola fish samples were collected from Hatir Pool Bazar (sample-1), Polashi Bazar (sample-2), Kaptan Baza (sample-3), Uttara (sample-5) of Dhaka and Joydebpur Bazar (sample-4), Shalna Bazar (sample-6), Tongi Bazar (sample-7) of Gazipur and kept in a covered jar and transferred immediately to the laboratory.

All the samples were extracted and cleaned up following procedure described in Section 2.6.1 and 2.6.3 (Page-33, 35).[39]

3.3 Analysis of DDTs

The clean up samples were analyzed by GC-ECD and the residual DDTs were identified and quantified.

3.4 Fat content

The range of fat contents were found to be 12-54, 43-78, and 50-293 mg/g ( fresh weight basis) in body, head and gut of mola fish samples, respectively. It was found that gut contained higher amount of fats. The fat content in body, head and gut of mola fish samples were represented in Table-2.4, 2.5, 2.6 (Page-42, 43,and 44 ) and Fig: 3.1, 3.2 and 3.3.

Figure:3.1 Fat content in body of different mola fish samples

Figure:3.2 Fat content in head of different mola fish samples

Figure:3.3 Fat content in gut of different mola fish samples

3.5 Standard calibration curve

Standard calibration curves were obtained by injecting 5 different concentrations (0.5, 0.25, 0.125, 0.0625 and 0.0312 ppm ) of standard pesticides into the GC-ECD. The standard calibration curves for DDE, DDD and DDT are given in Fig: 2.2, 2.3 and 2.4 (Page-45, 46 and 47).

3.6 LOD and LOQ

By analyzing the peak areas of standard solutions, the detection limit was found to be 0.39, 0.39 and 1.56 ng/g for DDE, DDD and DDT, respectively. The limit of quantification was found to be 1.36, 1.36 and 4.89 ng/g for DDE, DDD and DDT, respectively.

3.7 Recovery experiment

Recovery experiments of all samples were done for DDTs to calculate the accuracy of the determination. The values were found to be (70-120) % which is acceptable according standard methodology.

3.8 Residual DDTs in mola fish samples

DDTs in body of mola fish

The residual DDTs found in body of mola fish samples, expressed in ppb (ng/g) are presented in Table: 3.1 and Figure 3.4.

Table-3.1: DDTs in the body of mola fish samples

Sample ID Concentration Ratio

(DDT/DDTs)

DDE (ppb) DDD (ppb) DDT(ppb) DDTs (ppb)?
BH(1) 1.42 bql bql 1.42 _
BP(2) 3.15 1.39 8.47 13.01 0.651
BK(3) bql bql bql bql _
BJ(4) 3.99 2.27 6.96 13.22 0.526
BU(5) 1.63 1.67 15.4 18.7 0.824
BS(6) 2.49 bql 10.4 12.89 0.807
BT(7) 3.21 2.34 4.97 10.52 0.472
Mean 2.27 1.09 6.53 9.97 0.655
Max. 3.99 2.34 15.4 18.70 0.376
Min. 1.42 1.39 4.97 1.42 _

*(DDE+DDD+DDT)

bdl =below quantification level

(In our experiment bql ? 1.36 ppb for DDE and DDD, 4.89 ppb for DDT

*Replicate analysis was done for each sample

The experimental data showed that out of seven samples DDE, DDD and DDT were found in four samples. The range of DDE, DDD and DDT were found to be 1.42-3.99, 1.39-2.34 and 4.97-15.4 ppb, respectively.

Figure:3.4 Comparison of DDD, DDE and DDT in body of the different mola fish samples

The total DDTs were calculated and min, max, mean values were found as 1.42, 13.22 and 9.97 ppb, respectively. The ratio of DDT/DDTs was also calculated. From the ratio (>0.1) it was observed that contamination of DDT was recent and on going.

DDTs in head of mola fish

The residual DDTs in head of mola fish samples, expressed in ppb (ng/g), are presented in Table: 3.2 and Figure 3.5. The experimental data showed that out of seven samples DDE, DDD and DDT were found in five samples. The range of DDE, DDD and DDT were found to be 1.43-35.80, 1.39-16.84 and 4.93-14.49 ppb, respectively.

Table-3.2: DDTs in the head of mola fish samples

Sample ID Concentration Ratio

(DDT/DDTs)

DDE(ppb) DDD(ppb) DDT(ppb) DDTs(ppb)?
HH(1) 1.43 1.39 bql 2.82 _
HP(2) 4.45 2.19 4.93 11.57 0.426
HK(3) bdl bql bql bql _
HJ(4) 11.04 6.89 7.63 25.56 0.298
HU(5) 22.88 2.44 14.49 39.81 0.364
HS(6) 35.80 16.84 4.49 57.13 0.079
HT(7) 5.42 bql 8.25 13.67 0.604
Mean 11.57 4.25 5.53 24.56 0.225
Max. 35.80 16.84 14.49 57.13 0.254
Min. 1.43 1.39 4.93. 2.82 1.748

*(DDE+DDD+DDT)

bql =below quantification level

(In our experiment bql ? 1.36 ppb for DDE and DDD, 4.89 ppb for DDT)

*Replicate analysis was done for each of the samples

The total DDTs were calculated, and min, max and mean values were found as 2.82, 57.13 and 24.86 ppb, respectively. From the ratio of DDT/DDTs it was observed that contamination of fish is recent past and on going.

Figure:3.5 Comparison of DDD, DDE and DDT in head of the different mola fish samples

DDTs in gut of mola fish

The residual DDTs in gut of mola fish samples, expressed in ppb (ng/g), are presented in Table: 3.3 and Figure 3.6. The experimental data showed that out of seven samples DDE, DDD and DDT were found in four samples. The range of DDE, DDD and DDT were found to be 4.33-108.37, 2.83-9.56 and 4.93-49.67 ppb respectively.

Table-3.3: DDTs in the gut of mola fish sample

Sample ID Concentration Ratio

(DDT/DDTs)

DDE(ppb) DDD(ppb) DDT(ppb) DDTs*(ppmb
GH(1) 4.33 2.99 4.93 12.25 0.402
GP(2) 5.62 3.27 bql 8.89 _
GK(3) 82.20 bql 22.88 105.08 0.218
GJ(4) 18.58 bql 17.23 35.81 0.481
GU(5) 9.91 3.46 5.38 18.75 0.287
GS(6) 108.37 9.56 49.67 167.6 0.298
GT(7) 17.82 2.83 5.39 25.79 0.209
Mean 35.26 3.159 15.07 53.45 0.282
Max. 108.37 9.56 49.67 167.6 0.296
Min. 4.33 2.83 4.93 8.89 0.555

*(DDE+DDD+DDT)

bql =below quantification level

(In our experiment bql ? 1.36 ppb for DDE and DDD, 4.89 ppb for DDT)

*Replicate analysis was done for each of the samples

The total DDTs were calculated and min, max, mean values were found as 8.89, 167.6 and 53.45 ppb respectively. From the ratio of DDT/DDTs it was observed that contamination of DDTs was recent past and on going.

Figure :3.6: Comparison of DDD, DDE and DDT in gut of the different mola fish samples.

3.9 Discussion

The range of total DDT in body was found to be 1.43-13.22 ppb (ng/g f.w.) The higher amount of DDTs was found in mola fish collected from Joydebpur bazaar (18.70 ppb) and the lowest amount was found in the sample collected from Hatir pool bazaar (1.42 ppb).

The range of total DDT content in the head of all the mola fish samples was found as

2.82-57.13 ng/g f.w. The higher amount of DDT was found in sample collected from Salna bazaar (57.13 ppb) and the lowest amount of total DDT was found in the sample collected from Hatir pool bazaar(bql).

The range of total DDT in the gut of seven mola fish samples was found as 8.89-167.6 ng/g f.w. The higher amount of DDT was found in sample collected from Salna bazaar (167.6 ppb) and the lowest amount of total DDT was found in the sample collected from Palashi bazaar (8.89 ppb). The comparison of DDT, DDE and DDD in body, head and gut of different samples is shown in the Figure-3.7.

Figure:3.7 Comparison of DDTS in different mola fish samples

Earlier study conducted in our laboratory reported that DDTs were also present in different parts of mola fish which were in the range 5-11, 5-8 and 4-5 ppb for DDE, DDD and DDT, respectively in body of mola fish. The residual DDE, DDD and DDT were found in the range 7-15, 9-26 and 12-210 ppb, respectively in head while the range were 14-81, 28-64 and 13-43 ppb in gut. [41]

Out of three different parts of mola fish samples, higher amount of DDTs was found in head and gut. From our studies we observed that head (78 mg/g) and gut (293.5 mg/g) contained higher amount of fat. As DDTs are less water soluble and it can accumulate to fatty tissue of living organism, the higher amount of DDTs were found in head and gut of mola fish. From the properties of DDTs, they can bioaccumulate through the food chain to the human body.[42]

However, the residual DDTs are below MRL value of DDTs in fish (5 mg/kg).[43] From our present study it seems that mola fish contains lower amount of DDTs than MRL value. But continuous consumption of mola fish may accumulate DDTs in our body. As out of three parts body contains less fat and accummulate less DDTs, it is better to eat body than other parts of mola fish. From the ratio of DDT/DDTs it was observed that exposure of DDTs in mola fish is recent past and on going.

3.10 Conclusion

Seven mola fish samples were collected from different markets of Dhaka and Gazipur Body, head and gut of mola fish were separated and fat content were determined. These three parts were also analyzed for the presence of DDTs.The range of fat contents were found to be 12-54, 43-78, and 50-293 mg/g ( fresh weight basis) in body, head and gut of mola fish samples, respectively. It was found that gut and head contained higher amount of fats.

The range of total DDT content in the head of all the mola fish samples was found as

2.82-57.13 ng/g f.w. The range of total DDT in body was found to be 1.43-13.22 ppb (ng/g f.w). The range of total DDT in the gut of seven mola fish samples was found as 8.89-167.6 ng/g f.w.) Out of three different parts of mola fish samples, higher amount of DDTs was found in head and gut.

The amount is quite below the MRL value (5.0 ppm).[43] But continuous consumption of mola fish may cause more accumulation of DDTs in human body. The presence of DDTs signifies that DDT is still being used.