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This is to certify that the

thesis entitled

:PESTIC‘JBE QESllsUE ANALYSES (N ‘FR’ESHNKTEK
Emu 0%? MAIN LAKE ,11TA ,IBfiBANIN\aER\'R

presentedby
KOFFl Kopsawmu esouo

has been accepted towards fulfillment
of the requirements for

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7724M» W 13‘?in

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PESTICIDE RESIDUE ANALYSES IN FRESWATER FISH
OF MAIN LAKE, IITA, IBADAN, NIGERIA

BY

Koffi Kobenan Bouo

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Entomology
Pesticide Research Center

1986

 

ABSTRACT

PESTICIDE RESIDUE ANALYSES IN FRESHWATER FISH
OF MAIN LAKE, IITA, IBADAN, NIGERIA

BY

Koffi Kobenan Bouo

Some organochlorine and organophosphorus pesticides
were monitered in fish originated from Nigeria. Samples for
organochlorine pesticides were mixed with Sodium Sulfate and
blended with petroleum ether. A portion of the blend was
placed on a Florisil column and compounds were eluted with
mixtures containing 6 and 15% ethyl ether in petroleum
ether. Gas-liquid chromatography with electron capture de-
tection was used for determination of residues. DDT, Linda-
ne, Aldrin, Endosulfan, and Methoxychlor were found in all
samples at concentrations ranging from trace to 0.593 ppm.
Samples for organophosphorus (OP) residues were blended with
acetonitrile in lieu of petroleum ether. The blend was
cleaned up through hexane/acetonitrile partitioning. Gas-
liquid chromatography with flame photometric detection was
used for residue analyses. Only trace to 0.220 ppm of
Malaoxon was found in some samples. No other OP residues
were detected. Data are intended to provide an entry point
for future assessment of any change in pesticide exposure

levels in this lake.

ACKNOWLEDGEMENTS

I would like to record my sincere gratitude to my major
professor, Dr. Matthew Zabik, for giving me the opportunity
to work in his laboratory and for his guidance in the course
of this research project, in thesis preparation and through-
out my overall stay at Michigan State University (MSU).

Special thanks to Drs. R. Hoopingarner and J. Giesy
for serving on my degree committee. Thanks also to Drs.
Leavitt and E. Tukahirwa, Bob Kon, Bob Scheutz, Nelson
Herron, Jay Gooch, Susan Erhardt, Holly Fortnum, Ines Toro,
and Kim Hai-Dong for what we have shared together in the
laboratory.

I would also like to deeply thank the Government of The
Ivory Coast (Ministry of Agriculture), together with the
African-American Institute (AFGRAD) for their financial sup—
port. Grateful acknowledgement is extended to Ms. Elisabeth
Ward who made it possible for me to transfer from West Texas
State University to MSU where I have fully achieved my
academic goals.

Special thanks to everyone of my family, particularly
to Dr. Yao Nguettia René for his eternal advice and support.
I will be grateful to you all.

I finally extend my appreciation to all my special
friends; Lisa D. Yelder, Rachel Malaika, Patrice Koné, Gamal
Khedr, and Petros Charalambous for all that we have shared

together.

ii

TABLE OF CONTENTS

CONTENT page

ACKNOWLEDGEMENTS.... ........ . ............... . ............. ii
LIST OF TABLES O O 000000000000 O O O O O O O O O O O O O O O O O O O O O ......... V
LIST OF FIGURES.. ........... ... ........................... Vi
I. INTRODUCTION....... .................................. 1
II. ANALYTICAL METHODS .......................... . ........ 7
A. MATERIALS .................................... 7
1. Collection methods ........................ 7
2. Sample preparation .......... . ............. 7
3. Glassware preparation ..................... 7
4. Reagents......... ......................... 9
a O SOlvents O O O O O O O O O O ....... O ..... O ....... 9
b. Chemicals .............................. 9
c. Miscellaneous items ...... . ............. 10
B. ANALYTICAL PROCEDURES. ....................... 10
1. Organochlorine (0C) pesticides ............ 10
a. Extraction ............................. 10

b. Florisil chromatographic
column preparation and

clean—up.... ..... . ..................... 10

c. Quantitation ........................... 13

2. Organophoaphorus (OP) pesticides .......... 16
a. Extraction ............................. 16

b. Clean-up procedure ..................... 19

c. Quantitation...... ..................... l9

3. Fortification/Recovery .............. . ...24

iii

TABLE fl CONTENTS (continued)

 

CONTENT Page
III. RESULTS AND DISCUSSION ............................... 26

A. RESULTS......................................26

B. DISCUSSION........... ........................ 29
IV. CONCLUSIONS .......................................... 36
APPENDIX........................ ....... ...................38
LIST OF REFERENCES ................. . ..... .................44

iv

TABLE

 

LIST OF TABLES

Page
Fish from Main Lake (IITA) sampled for
residue analysis.......................... ........ 8
List of selected pesticides studied...............11
Recovery of selected organochlorine and
organophosphorus pesticides studied ............... 25
Average and range of concentration
(mg/kg wet weight) of organochlorine
pesticides found in samples of fish
collected from Main Lake (IITA) in Ibadan ..... ....27
Average and range of concentration
(mg/kg wet weight) of organophosphorus
pesticides found in samples of fish
collected from Main Lake (IITA) in Ibadan ....... ..30
List of pesticides in use and/or in stock
at the International Institute of Tropical
Agriculture (IITA) ...................... . ......... 38

FIGURE

LIST OF FIGURES

page
Extraction scheme for organochlorine

pesticides .................. .. ................ ....14
Clean-up scheme for organochlorine
pesticides................ ........................ 15
Reconstructed chromatogram of a standard

mixture of chlorinated pesticides studied.........17
Reconstructed chromatogram of a sample of

fish analyzed for residues of chlorinated
pesticides........ ..... ......... ..... . ............ 18
Analytical scheme for organophosphate

pesticides studied ................................ 20
Reconstructed chromatogram of a standard

mixture of organophosphorus pesticides

studied...... .................... . ................ 21
Reconstructed chromatogram of a sample of

fish analyzed for residues of organophophorus
pesticides ........................................ 22

vi

 

I. INTRODUCTION

Agriculture has evolved throughout the world, espe~
cially in developed nations, as a result of innovative de—
velopments of many types. For instances, diverse and spe-
cialized machinery, development of productive varieties of
plants, development and use of chemical fertilizers, and
discovery and use of pesticides, to mention just a few, have
helped to maintain adequate food supplies in many parts of
the globe. In contrast to the developed nations, many deve-
loping countries still suffer from low productivity. In the
same countries, excessive loss of food crops to insects and
other destructive pests leads obviously to starvation. In
all these countries use of pesticides remains one of the
most powerful and dependable tools available for controlling
these pests. These chemicals are more effective, economical,
and adaptable for use in a variety of situations than any
other proved tools for controlling pest populations at sub—

economical levels (Newson et al.,1976).

 

As man has developed machinery and pesticides to sus—
tain and increase productivity he has, at the same time,
developed source of environmental pollution that has had
adverse effects on nontarget plants and animals, including
humans, our waterways (Stickel,1968) beside other related
problems such as increasing number of resistant pest species
(Smith,1976; Brown,1977,1978; Croft,l978). On the other

hand, intensive use of pesticides in agriculture today has

 

 

led to increasing awareness of the problem of safeguarding
the consumer and the environment.

Upon release in the environment a chemical may be
metabolized by living organisms, be transformed through
chemical or photochemical reactions, or persists unaltered.
In some instances degradation or transformation results in

toxic products (Menzie,1972; Crosby,1973; Goring gt al.,

 

1975). There are several properties of pesticides that con—
tribute to their behavior as pollutants. Among these are
toxicity, stability, solubility, and adsorptivity.

Different types of pesticides vary greatly in their
toxicity to animals and plants. Insecticides, for example,
are selected for their toxicity to insects whereas herbi-
cides are selected for their toxicity to weeds.

Stability or persistence implies a chemical charac-
teristic giving the products long live in soil and aquatic
environments, and animal and plant tissues. They are not
readily broken down by microorganisms, enzymes, heat or
ultraviolet light. From the insecticidal viewpoint these are
good characteristics. From the environmental viewpoint they
are not. DDT and other chlorinated hydrocarbons are among
the most noteworthy examples for their persistence. Their
stability combined with their solubility in lipids account
for their bioaccumulation and biomagnification. In contrast
to the lipid—soluble chemicals, the water—soluble or polar

compounds generally are excreted by animals and tend to

 

 

remain in the aqueous medium where they are readily availa—
ble to attack by microorganisms.

Adsorption or binding of a chemical to soil colloids or
other micellar components in the environment tends to
decrease its availability to plants and animals, including
microorganisms and to subsequently reduce it decomposition.

In view of the importance of the environmental quality
control many countries have introduced rigid legislation
requiring detailed examination of all kinds of potential ha—
zards before a new agrochemical can be approved for specific
usage. In the United States, for example, the Environmental
Protection Agency (EPA) is basically the primary regulatory
institution to take such measures.

Residues, hazards, and legal problems are all functions
of the overall pesticide load placed on the agroecosystem.
The significance of these problems is at best poorly under-
stood on a worldwide basis because developing countries do
not have qualified personel and the technological systems
necessary to monitor pesticide residue levels, distribution,
and degradation in the environment.

The aquatic environment in particular serves as a
reservoir for tremendous quantities of foreign organic
chemicals, or xenobiotics. These compounds, many of which
are toxic to both aquatic and mammalian species (Matsumura,

1975; Cin g; al.,1982), enter our waterways through various

 

routes. Aquatic organisms may be exposed to xenobiotics,

including pesticides by intentional contamination as in the

 

 

case of sewage effluents, hydrocarbons, lampricides,

molluscides, and mosquitoe larvicides (Manda et al.,1974;

 

Cooper,1978; Argaman,1978). Unintentional contamination may
result from run—off of pesticides, industrial effluents,
hydrocarbons, and other waste substances into the aquatic
habitat (Keith,1974,1975; Kanazawa,1975; Carter,1978;
Haller,1978).

Cases of water contamination with organochlorine
pesticides or industrial chemicals were much in the news
during the 1960's and 1970's. The result has often been an
appearence of persistent contaminants in the exposed aquatic
life. Such cases of alleged environmental pollution include
PCBs in the Hudson River and the Great Lakes, Mirex in the
Great Lakes, and Kepone in the James River. The direct con—
sequences that one can infer from this type of pollution
are: 1) that exposed aquatic organisms (e.g., fish) may
express adverse biological effects which can bring about

death (Sheila gt al.,1982) and 2) when some of these sub-

 

stances (e.g., organochlorine insecticides) are incorporated
by fish (or plant or animal) into the food chain they pass
along it and accumulate in the highest predator in the
chain, so that a lethal concentration may be obtained at a
level several thousand times that found in the actual water

(Young gt al.,1979; Fry and Toone,1981). Therefore, aquatic

 

animals consumed as foodstuff may represent a potential
source of human exposure to toxic xenobiotics, including

carcinogens and mutagens.

 

 

Since man heavily depends on animal proteins (e.g.,
fish) fate of these xenobiotics in aquatic species is of
importance. Hence the concern about Main Lake at the Inter-
national Institute of Tropical Agriculture (IITA) at Ibadan,
Nigeria. IITA is one of the major links in a worldwide net-
work of agricultural research and training centers (IITA
Research Highlights,1983).

The importance of Main Lake for fish production, water
supplies for both human needs and agricultural purposes
(e.g., irrigation) has greatly increased over the years with
growing populations. In addition, since outflow of this lake
is limited (surrounded by agricultural lands) chemical dis—
charges can be very persistent.

Currently there is much concern over the environmental
quality of this lake with regard to its fish proteins and
drinking water along with general public health. Consequent-
ly IITA has requested an evaluation on how much Main Lake is
"polluted" through biological matrices (fish) after several
years of expanding use of pesticides on nearby farmlands.

Organochlorine (OC) insecticides were given priority in
our study because of their well known environmental persis-
tence and high toxicity to marine organisms (Goldberg,1975;
Portmann,1975). Moreover, OC pesticides, even though discon—
tinued in use in some nations (e.g., DDT banned in the USA),
are still being used and will probably continue to be used
for some time in developing countries, further increase the

need to study these chemicals.

 

 

Of equal importance to this investigation were the
organophosphorus (OP) insecticides, most of which are known
to be more toxic and less persistent than the OCs
(Kanazawa,1975; Matsumura,1975), for they have also been
used among other classes of chemicals at the IITA.

The purpose of this investigation was :

1. to determine the presence and magnitude of
pesticide residues in fish of Main Lake;

2. to subsequently measure regional pollution
believed to be caused by agricultural
discharges into this lake; and,

3. to establish an initial baseline for comparison
with future work for this region of Nigeria.

Our initial studies, which are reported here, describe
the concentration levels and significance of nine selected
pesticides in twelve fish species of Main Lake. At present,
no comprehensive trace study in fish has been conducted at

the IITA.

 

 

 

II. ANALYTICAL METHODS
18. IIATERIALS
1. Collection methods
Details on methods for fish collection are
lacking. However, whole fish belonging to twelve different
species (Table 1) were brought to our laboratory for trace
analyses following capture. The original samples were then
kept frozen (—20 0C) until analysis.
2. Sample preparation
In the laboratory each whole fish was
considered as one sample. Each fish was allowed to thaw,
rinsed with tap water, shaken dry, scaled off, and weighted.
Then, fish was individually ground in an industrial type
blendor (Model CB-5, Waring Blendor, Waring Products Co.,
Winted, Conn.) until a homogenous puree was obtained. The
finely ground sample was subsampled into widemouth-screw-cap
bottles with aluminum foil-lined caps. Every subsample was
properly labelled and stored in freezer at -20 0C until
analysis.
3. Glassware preparation
All glassware (separatory funnels, beakers,
flasks, funnels, Teflon seals, and chromatographic tubes)
were thoroughly washed sudy in hot water, rinsed out several
times with tap water, then distilled water and, finally,
with acetone (plus an additional appropriate solvent if

necessary, for used glassware only -"like dissolves like")

 

 

 

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to get rid of any interfering contaminants. The glassware

was then heated overnight in a furnacle at 450 0C before

usage. Teflon seals, on the other hand, were dried out in an

oven at less than 100 0C to prevent degradation of the

layers.

4. Reagents

a.

b.

Solvents
i) All solvents; petroleum ether(PE), ethyl
ether(EE), hexane, acetone, and acetonitrile
were pesticide grade, glass distilled, and
used as received.
ii) Solvent mixtures
* Mixture A : 94% PE - 6% EE
* Mixture B : 85% PE - 15% EE
Chemicals
1) Sodium Sulfate - NaZSO4 (ACS) granular, an-
hydrous, reagent grade, and free of inter-
ference with the electron capture detector.
ii) Florisil - PR grade, 60-100 mesh, activated
in oven at 135 0C for 48 hrs. After cooling
in a dessicator at room temperature, the
activated Florisil was stored in glass con-
tainers with foil-lined screw caps. Enough
Florisil from the same batch was submitted

to the same treatment for use during the

entire work.

 

 

10

0. Miscellaneous items
i) Glass wool (Pyrex) - free of interference
with the electron capture detector (ECD).
ii) Glass filter 17GB or equivalent was used.
iii) Reference chemical standards - all pesti-
cides (Table 2) were obtained from EPA,
Research Triangle Park, N.C..
13. ZXNALYTICAL IPROCEDURES
1. Organochlorine (OC) pesticides
a. Extraction
Fish sample (thoroughly ground and mixed)

(10g) was mixed with Na 804 (10g) and blended, for 1-2 minu-

2
tes, with petroleum ether (PE) (50 ml) in Sorvall Omni—
Mixer. Following centrifugation at ca 2000 rpm for 1-2 minu-
tes, the PE extract was decanted through a Na2804 layer (in
order to remove the excess water) into a 125 ml volumetric
flask. Two additional extractions were carried out as in
above using 50 ml and 35 ml of PE, respectively. The total
blend volume was diluted to 125 ml with a portion of PE be-
fore proceeding to the clean-up step. A 25 ml aliquot of the
blend was removed for gravimetric determination of per cent
fat. Another 25 ml aliquot was concentrated to approximately
2-3 ml on a rotary film evaporator for introduction onto a

clean-up column.

b. Florisil chromatographic column preparation and
clean-up

Pyrex columns (10 cm internal diameter(i.d.)

x 51 cm length (l) with Teflon stopcocks) were packed with

 

 

11

Table 2 - List of selected pesticides studied

 

 

 

Class/Common Name Molecular Formula
Organochlorine
pesticides
Lindane, BHC-gamma C6H6Cl6
isomer
Aldrin (HNDN) C12H8C16
Endosulfan I C9H6Cl6O3S
Endosulfan II C9H6Cl6O3S
'-
p,p DDT C14H9Cl5
.. I
Methoxychlor P1P C16H15Cl302
Organophosphate
pesticides
Malathion C10H1906PS
Malaoxon C H 0 PS
(Malathion oxygen 10 19 7
analog)
Monocrotophos C H NO P

 

12

4g of activated Florisil topped with a 2 cm NaZSO4 layer.
This was achieved by gently taping the chromatographic tube.
Each tube or column was washed with 20-25 ml of PE. The
column was not allowed to dry at any time in the procedure.

The concentrate (residues) was transfered
into the column using disposable Pasteur pipets. Then, the
container was washed out with 1 ml of PE and wash was added
to the column. Compounds were eluted with Mixture A (35 ml
of 94% PE - 6% EE) for very non-polar pesticides such as
Aldrin, Lindane, DDT and analogs, and PCBs if any. Relative-
ly polar compounds like Dieldrin and Methoxychlor were elu-
ted with Mixture B (35 ml of 85% PE - 15% EE). All eluted
fractions were concentrated on a rotary film evaporator (or
under liquid Nitrogen) to appropriate volume for gas-liquid
chromatography (GLC) determination.

At this juncture it is essential to point out
some aspects on the variability in Florisil activity. Flori-
sil is a polar adsorbent known to have a large surface area,
which is the basis of its adsorption properties. Paul A.
Mills (1968) reported that the adsorption capacity of Flori-
sil varied from one batch to another due to varying Sodium
Sulfate content. Since then, several methods had been asses-
sed to rule out this constraint. Lauric acid method is one
commonly used example among major breakthroughs. In our stu—
dy, however, we bipassed this problem by simply standardi-
zing the chromatographic column with 15 ml of PE containing

10 ppm of the chemicals studied. Care was taken to keep the

 

13

Florisil activity rather constant by avoiding long exposure
of the treated Florisil to humidity.

These extraction and clean-up procedures
generally follow those set up by Ronald D. Erney (1983) but
with slight modifications. His methodology was developed as
a rapid and reliable screening procedure for pesticides and
PCBs in fish (collaborative study) and was proven to be ef-
ficient, less time-consuming, economic, and comparable to
the official methods for determination of residues (Erney,
1983).

Figures 1 & 2 summarize the experimental sec-
tion as described above.

c. Quantitation

A standard curve was constructed for each
pesticide from different concentration levels of standard
solutions following injections of appropriate volume into
the GLC-ECD.

A Tracor 560 Gas Chromatograph equipped with
a discharge 63Ni electron capture detector was used for the
analyses. It was fitted with a D81 fused Silica capillary
column (30 m l. x 0.25 mm i.d.) with 25 micron liquid phase
thickness and was operated at a column (oven) temperature of
270 OC and a 30 ml/min. Nitrogen (99.995% purity) flow rate.
The injection port temperature was 285 OC and the detector
temperature 275 0C. A SP4270 Spectra Physics Integrator was

used for recording.

14

Figure 1 - Extraction scheme for organochlorine pesticides

10g of fish sample
(in metal cup)

1. 10g of Na SO

2. mix with Stigring rod

3. let setlle 20 min.; mix again

4. 50 ml extraction

5. blend 1-2 min.

6. centrifuge 1-2 min. at 1000 rpm

7. decant PE extract thru Na SO
layer (used glass wool plag in
funnel) into 125 ml vol. flask

 

Sample PE extractl

8. repeat steps 2-7 1

 

PE extract2
Sample
9. mix again

10. add 35 ml PE :
11. repeat steps 5-7 x

 

 

PE extract2

 

 

 

Sample Combined PE extracts
(discard) l
:1. dilute to 125ml
with PE
25 ml aliquot 25 ml aliquot
1. transfer into scaled flask 1. concentrate
2. evaporate to near dryness to 2-3 ml
3. weight flask
Determine % fat Residues

(for clean-up)

15
Figure 2 - Clean-up scheme for organochlorine pesticides

Residues

1. transfer residues and wash of
container into column

49 Florisil column
(+ 2 cm Na2804 on top)
1. mark tube 1 cm above Na SO4
2. follow standardization as
outlined in Text
3. elute with 35 ml Mixture A

: 4. continue

.: elution

r with 35 m1
‘: Mixture B

i

 

6% BE in PE: 15% BE in PE:
non-polar compounds; polar compounds;
DDT Methoxychlor
Lindane... Dieldrin...

(concentrate) (concentrate)

 

+GLC/ECD+

 

16

Standard mixtures were injected at the

beginning of each run, after every three samples, and at

the end of the run. Figures 3 & 4 illustrate reconstructed

chromatograms of a standard mixture and a sample.

Quantitations were based upon peak heights

(or areas) and the concentration levels for each compound

were determined on the basis of wet weight of fish according

to the following universal equation :

R

where a

e

------- (eq. #1)

nanograms of pesticide represented by the
standard peak

height (or area) of sample

height (or area) of standard peak

grams of original sample

residue concentration in parts per million
or billion (ppm or ppb)

Dilution Factor derived from eq. #2 below:

ml of extracting solvent x volume of final extract*

aliquot taken of original extract (ml) x ul injected

* This value is in ul for ppb and in ml for ppm

2. Organophosphorus (OP) pesticides

a. Extraction

After adding 10g of Na 80 and 100 ml (or 2 x

2 4

50 ml) of acetonitrile, the fish sample (109) was blended in

Sorvall Omni-Mixer, for 1-2 minutes, and filtered on filter

papers 17G3 (or Whatman glass microfilter-GF/C). The extract

17

Figure 3 - Reconstructed chromatogram of a standard mixture of
chlorinated pesticides studied

It“!

Solvent

 

 

Lindane

"-E.

‘1‘
Ill
..

 
 

 

 

Gas chromatographic conditions:

Column — DB1 fused Silica, 25 micron liquid phase
thickness (capillary column)

Oven - 270 C

Detector — 63Ni EC, 275 C

Carrier - N2, 30 ml/min.

Recorder — SP427O Integrator

C.
'H
L4
"('3
....)
<

'33

rn

I

III

‘7‘!

T
J.

  

L4
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g H a .c
M H Q U
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:1 C‘. X
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'U H u
a 3 m
m 8 III 2
"U .
c‘.
.. m r .1 I33 ' ‘4
' U‘J

 

 

 

 

 

 

 

 

 

 

 

Note : Numbers represent retention times (minutes) for

W .

standard chemicals

18

Figure 4 - Reconstructed chromatogram of a sample of fish analyzed
for residues of chlorinated pesticides

Gas chromatographic conditions:
Column — DB1 fused Silica, 25 micron liquid phase
thickness (capillary column)

 
    

 

 

  
   

Oven — 270 C
Detector — 63N1 EC, 275 0
Carrier — N2, 30 ml/min. F
.fi. Recorder - SP4270 Integrator
W)
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-M
in
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Ti
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a
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rt H
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Note : Numbers represent retention times (minutes) for components
in injected sample

—..'

w

.7: W —

Methoxychlor

.... ....
......k

_ —-—.*‘

 

 

....)

 

..-- -.——.—.—— .

 

 

19

was concentrated below 50 0C on a rotary film evaporator for
clean-up.
b. Clean—up procedure

The concentrate (residues) was dissolved in
hexane (25 ml) and transfered into a 100 ml separatory fun-
nel, and extracted twice with each 25 ml of acetonitrile.
The acetonitrile extracts were combined, and concentrated as
in above and dissolved again in appropriate volume of
acetone for GLC determination.

These extraction and clean-up procedures
strictly followed those developed by Jun Kanazawa (1975) and
were used as described with no modifications. The experimen-
tal section is given in Figure 5.

c. Quantitation

As in the case of the organochlorine (0C)
pesticides a standard curve was also obtained for every OP
compound. In a similar manner to the OCs, samples and stan-
dard solutions were injected into the GLC as well. Figures 6
& 7 show reconstructed chromatograms of a standard mixture
and a sample.

OP residues were determined on a Beckman
GC-65 gas-liquid Chromatograph equipped with a flame photo-
metric detector in the phosphorus mode. Analyses were per-
formed at the following operating conditions

- Column : Pyrex, 6 ft. (1.83 m) x 1/18 in.
(1.59 mm) i.d. packed with 4% SE 30

+ 6% 0V 210 on 80/100 Chromosorb W-HP

20
Figure 5 - Analytical scheme for organophosphate pesticides

10g of fish sample

1
:1. 10g Na so

:2. 50 ml acetonitrile extr.
:3. blend 1-2 min.

(4. filtration

1

 

 

 

 

Filtrate1 Sample
5. repeat steps
2-4
Filtrate2
Combined filtrates Sample

(discard)

1. concentrate
2. dissolve residues in 25 m1 hexane

Transfer into 100 ml sep. funnel

1. add 25 ml acetonitrile
2. lst acetonitrile/hexane(1:1) partition

 

Hexane fraction Acetonitrile fraction
J
:3. 2nd acetonitrile/

hexane(l:1) partition

 

Acetonitrile phase

1
1
1
1
1
4
1

 

l

Hexane fraction Combined acetonitrile extracts

(discard)
1. concentrate

2. dissolve in acetone

GLC/FPD

21

Figure 6 - Reconstructed chromatogram of a standard mixture of
organophosphorus pesticides studied

Gas chromatographic conditions:
Column - 4% SE30 + 6% OVZlO on Chrom.
Oven - 140 C:

Detector - FPD (P mode), 285 C
Carrier - N2, 30 ml/min.
Recorder chart speed - 0.2"/min.

Solvent
alathion

Malaoxon

 

 

 

AU

0 0 O
i i 5 Time (min.)

Note : Monocrotophos is not chromatographed because it has about
the same retention time as Malathion

22

Figure 7 - Reconstructed chromatogram of a sample of fish
analyzed for residues of organophosphorus pesticides

Gas chromatographic conditions:
Column — 4% SE30 + 6% OV210 on Chrom.
Oven — 140 C

Detector — FPD (P mode), 285 C
Carrier — N2, 30 ml/min.

Recorder chart speed - O.2"/min.

Solvent

::=== Malaoxon

 

 

 

l I 1.1

d_. lb ‘20 Time (min.)

23

- Detector temperature : 285 oC

- Column (oven) temperature : 140 0C

- Injection port temperature : 280 oC

- Nitrogen flow rate : 30 ml/min.

- Air flow rate : 115 ml/min.

- Helium flow rate : 120 ml/min.

- Recorder chart speed : 0.2 in./min. (0.5

cm/min.)
Quantitations and concentration levels of

each compound were performed under the same conditions as
outlined for the 0C pesticides. Therefore, the residue equa-

tion (eq. #1) would be applicable to the OPS accordingly,

that is,

R = ------- (eq. #1)

where only "e" spells different, the other parameters
remaining the same. In this particular case the Dilution
Factor (e) is given by equation #3 as follows :
volume of final extract (ul or ml)
ul injected

Since the final extract (concentrate) contained the
entire original sample (no aliquot was taken), the values
for the "m1 of extracting solvent" and the "aliquot taken of

original extract" in eq. #2 would cancel out to give "e" as

in eq. #3 above.

24

3. Fortification/Recovery
To evaluate performence of the foregoing
analytical procedures, known amounts of different concen-
tration levels of pesticides to be determined (2, 5, and 20
times the limits of detection) were added to samples prior
to extraction. This process is referred to as fortification.
Recoveries (or per cent recovery) were
determined at fortification levels ranging from 0.01 to 3.0
ppm for the OC pesticides and from 1.0 to 15.0 ppm for OP
chemicals before the blending operations, and the fortified
samples were then carried through the above procedures . Per
cent recovery was derived from equation #4 below :
amount of pesticide obtained
% recovery = ---------------------------- x100 (eq. #4)
amount of pesticide added
Table 3 gives % recovery for every compound
of interest. Per cent recovery ranging from 79% to 99.8% are
indication of good and dependable analytical procedures

regardless of modifications undertaken in our study.

 

25

Table 3 - Recovery of selected organochlorine and
organophosphorus pesticides studied

 

Compound Average percent recovery (%)
Lindane 99.8
Aldrin 89.8
Endosulfan I 88.5
Endosulfan II 85.7
p,p'-DDT 90.0
Methoxychlor 79.3
Malathion 91.2
Malaoxon 95.6

Monocrotophos 85.2

 

26

III. RESULTS AND DISCUSSION
28. IRESULTS
Residue levels in fish are expressed as mg/kg wet
weight. All of the six organochlorine (OC) pesticides listed
in Table 2 were found in all samples of fish studied (Table
4). On no occasion was there any indication of polychlori- f
nated biphenyl (PCB) contamination. Residue concentrations
varied from trace to a maximum average of 0.593 mg/kg. DDT

and Lindane ranked high in the majority of the samples, the

 

main one being DDT in all species at average concentration
levels ranging from 0.143 to 0.593 mg/kg wet weight whereas
Lindane ranged from 0.443 down to 0.036 mg/kg wet weight.
Aldrin, Endosulfan I & II, and Methoxychlor had a highly
scattered distribution in all species with an overall
average concentration levels of 0.004 up to 0.114 mg/kg

wet weight. Among species the highest residue levels of

DDT (0.593 mg/kg) were detected in samples of a specimen

of Oreochromis niloticus (Nile Tilapia), those of Lindane
(0.443 mg/kg) and Methoxychlor (0.114 mg/kg) in samples of
Heterotis niloticus (Stone Head), and those of Aldrin (0.076
mg/kg) and Endosulfan I (0.133 mg/kg) in samples of
Auchenoqlanis occidentalis (Yellow Catfish) whereas those

of Endosulfan II (0.088 mg/kg) were found in samples of
Morqurops delicious. All in all, residues of DDT followed
by Lindane were constantly higher in samples of every single
fish species in comparison with concentration levels of

Methoxychlor, Aldrin, and the two isomers of Endosulfan,

27

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28

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0A0.0 m00.0 H.0H mficmamococos<
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A masseucoo v I «.0H9m9

 

29

which were relatively lower in the majority of fish
specimens.

Although each sample was screened for three
organophosphate (OP) pesticide residues, Malaoxon (Malathion
oxygen analog) was the only residue detected in some sam-
ples of fish at concentration levels ranging from trace to
a maximum average of 0.220 mg/kg wet weight (Table 5). Laggs
niloticus (Niger Perch) followed by Clarias lazera (African
Mudfish) carried the highest residues; 0.220 and 0.189
mg/kg, respectively. The tabulated results of the study
indicate the essential absence of any other OP pesticides,
including Malathion and Monocrotophos.

B. DISCUSSION

There must always be some ambiguity in the com-
parison of residue data from different species in the
absence of controlled experiments on their ability to
accumulate pesticides. In addition, since apparently no
other trace studies in fish of Main Lake have been con—
ducted and no detailed information on pesticide use pat-
terns at this location is available, it is difficult to
assess whether the scattered distribution of concentra—
tion levels reported here is common in this environment
or whether it is particularly due to species differences.
In an effort to explain the significance of the degree of
contamination of the organisms sampled, substancial infor-

mation available in the literature was primarily considered.

30

Table 5 — Average and range of concentration ( mg/kg wet weight ) of
organophosphorus pesticides found in samples of fish
collected from Main Lake ( IITA ) in Ibadan

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Species Malathion Malaoxon Monocrotophos

Oreochromis niloticus ND 0.026 ND
(ND—.034)

Tilapia guineensis ND » 0.013 ND
(ND—.016)

Tilapia zillii ND 0.005 ND
(ND—.011)

Sarotherodon galilaeus ND 0.006 ND
(ND—.009)

Lates niloticus ND 0.220 ND
(T-.305)

Chromidotilapia guntheri ND T ND
(ND-T)

Hepsetus odoe ND T ND
(ND—T)

Channa obscura ND 0.003 ND
(ND—.018)

Mormygrgps delicious ND 0.103 ND
(T—.114)

Clarias lazera ND 0.189 ND

' (T-.215)

Auchenoglanis occidentalis ND 0.087 ND
(T—.105)

Heterotis niloticus ND 0.120 ND
(T—.196)

 

 

 

Notes : ND — Non-detectable concentration level
I - Trace level
Sample size : 5 - 8

 

 

31

None of the twelve fish species analyzed con-
tained individual and average residue levels in excess of
the 5 ppm (mg/kg) tolerance level of DDT established by
the Food and Drug Administration (FDA), US Department of
Health, Education, and Welfare or Health and Welfare in
Canada. The relatively low concentrations of all the
chlorinated pesticides studied at individual and maximum
average residue levels less than 1.0 ppm or mg/kg wet
weight in all fish specimens probably are biologically
insignificant. However, DDT burdens ranging from 1.0 to
4.0 ppm if any could cause physiological stress and les-
sen reproductive capacity in fish populations (Butler gt
git, 1972). Many samples of freshwater fish were reported
to contain higher pesticide concentration of chlorinated
pesticide residues (mainly DDT and Dieldrin) than those
of commercial fish, most of which are of marine origine
(Hays,1975). Fish are able to take up all organochlorine
pesticides (DDT being the best example in this case) rapi-
dly and directly from water; the absorption occurs mainly
through the gills and does not depend on food. Although fish
can absorb DDT from that source also, two factors involved
in direct uptake from water are absorption and lipid parti-
tioning. Both factors are in the behavior of DDT and other
chlorinated pesticides. DDT for instance is not only
absorbed on the surface of the gills, living algae, and
particles of dead organisms but also on surfaces generally,

including those of silts (McKim gt al.,1974), which probably

 

32

reflects relatively high concentrations of this compound in
this study. It was reported elsewhere that fish were able to
accumulate materilal from the suspended materials
(Zitko,1974), however the mechanism by which uptake occurs
is not understood. Neely gt git (1974) and Kanazawa (1982)
reported that for many non—ionic organic compounds, includ-
ing several pesticides the bioconcentration factor (BCF)
increased as the molecular weights increased. According to
the same authors, the BCF from water by fish increases as
the solubility in water decreases, or as the 1-octanol-water
partition coefficient increases. Our data also support this
contention, at least for DDT residue levels. Furthermore,
the constency higher concentration levels for DDT and
Lindane in the majority of fish species probably were due to
differences in age, in species, and more likely to the high
fat content and the carnivorous behavior of some of these
specimens, which in turn lead to biomagnification in top-
order consumers. Because DDT is metabolized to DDD and DDE
(Menzie,1978) proportionately high concentrations of DDT in
fish suggest possible build-up and/or continuing inputs of
this material to the aquatic ecosystem (Aguillar,1984). Our
data support these observations. However, as Table 4 illus-
trates, our data sometimes refute the hypothesis that diffe-
rences in organochlorine pesticide residues between species
at a given site (Main Lake) are related to their differing
lipid levels. These results support one conclusion reported

by Schmitt et a1. (1981): lipid content alone does not

33

adequately explain differences in residue levels between
species at a given location.

The BHC isomers are relatively shorted-lived com-
pared with some organochlorine (0C) pesticides. The gamma
isomer, Lindane, is one of the 0C pesticides that has been
widely used at the IITA. It has a BCF of 50 - 900, depending
on species and environmental conditions, and thus does not
normally accumulate (Kanazawa,1978; Sugiriura gt git,1979).
Its occurence, at more than trace, therefore indicates
build-up and/or continuing inputs in the aquatic habitat.

Aldrin and the two isomers of Endosulfan , which
have relatively high BCF's and long half-lives in fish

samples (Clark gt al.,1983), were found at very low concen-

 

tration levels in the majority of fish species, but their
presence has no apparent correlation with their chemical
properties. Another consideration in determining fish body
burdens is the fact that the rate and frequency of use of
these chemicals in the Ibadan area (IITA) might be relati-
vely low compared with those of DDT and Lindane, which could
explain residue differences in fish. Aldrin which is metabo-
lized to Dieldrin (0.3 ppm tolerance guideline set by USFDA)
probably does not represent at this point in time any threat
to fish, wildlife, and the consumer. A similar argumentation
can be made on the behalf of the two isomers of Endosulfan.
On the basis of the information gathered
(Appendix), Methoxychlor has not been used at the IITA.

Its presence in all samples studied therefore suggests an

34

outside source of contamination. This may be attributed

to atmospheric transport into the lake or to the migratory
behavior of the majority of the specimens sampled, which
probably explains the subsequent uptake of this material.
Because Methoxychlor is shorted-lived and bioaccumulates
to a lesser degree than DDT and many other OC pesticides

(Kapoor gt al.,1973) the residue levels found in fish

 

probably reflect the continuing outside inputs of this
compound in the aquatic environment. The current con-
centration levels probably are biologically insignificant
and therefore should not be of concern to the consumer.

The almost non-detectable levels of organophos-
phate (OP) pesticide residues in this study, Malaoxon
(a Malathion metabolite) being sometimes the exception in
some samples, are consistent with observations made in many

other parts of the world (Spehar gt al.,1980; Reish gt

 

al.,1981; Chovelon gt al.,1984). This reflects the relative-
ly slow uptake and high metabolism/excretion rate of these

substances by aquatic organisms (McLeese gt al.,1979) which

 

in turn produce relatively low concentration factors. For
example, the 7 - 14 day concentration factor of Diazinon
varies from 18 - 206, depending on species (Kanazawa,1978).
Jun kanazawa (1975) reported that Malathion among other OP
compounds taken up by fish was metabolized rapidily, which
is also consistent with our observation. Although Malathion
was not directly detected, residues of Malaoxon found in

samples indicate that uptake followed by metabolism/excre-

35

tion of this material by fish had occured before the sampl-
ing time. Another important consideration in determining
fish body burdens is the fact that the rate and frequency
of OP pesticide usage at the IITA might be relatively low
compared with those of the OC pesticides. In addition, the
sultry tropical climate in Nigeria might have drastically
impacted on the effective life of pesticides, including the
CPS which degrade rather easily by hydrolysis. By large, the
OP pesticides are more toxic and less persistent than the
OCs and residue levels reported here probably are insigni-

ficant to biological systems.

36

IV. CONCLUSIONS

The results of this trace study in fish of Main Lake
(IITA) show basically that residues of organochlorine (OC)
pesticides (and the OPs to some extent) are present, though
at low concentrations, in the majority of common fish
species. Although it is generally the case that marine fish
are contaminated with OC compounds (Portmann,1975), there is
little evidence that levels currently found in most parts
of the world are of any significance to the fish. It is,
therefore, unlikely that the reported low residue levels
from Main Lake will have even a sublethal effect on healthy
animals. Animals forced to mobolize their lipid reserves
during periods of starvation may be an exception. The
significance of the latter circumstance is difficult to
evaluate and will certainly vary with species, season, and
such body burdens as diseases and parasites (Olafson,1978).
These residues have apparently originated from the areas of
intense pesticide application in the Ibadan region (IITA).
The natural processes of weathering are likely to result in
the transfer of pesticide residues to the main body of the
lake, especially during wet seasons, where they can
accumulate and persist. Since OC pesticides have high
ecological magnification values and low biodegradation
indices in a laboratory model ecosystem, compounds like DDT
accumulate in the tissues of levels 200 to 84,000 times
those found in the actual water (McKim, 1974). The water

treatment if any actually has no effect on the concentration

37

of DDT in the water (Sunshine, Z. Editor,1969). In other
words, OC pesticides (unlike the CPS) in water will be taken
up very rapidly by living organisms (Sodergren,1968) or are
either completely adsorbed by particulate matter (Keith and
Hunt,1966). Main Lake is the site of deposition (limited
outflow) of xenobiotics and suspended matter. Under these
circumstances, and in View of the continuous use of OC
pesticides in agriculture in this region, it is expected
that an appreciable build-up of residues with time will take
place in this lake. Increased contamination with residues is
certain to adversly affect the fish populations

(Holden,1965; Kanazawa,1975; Sheila gt al.,1982) and hence

 

endanger the plans for the development of a fisheries indus—
try in this vital area.

Effective management of toxic substances in the envi-
ronment requires a committment to long-term monitoring.
Consequently, in order to keep the situation (environmental
pollution) under control, it is essential that a system or
fate models for the continuous monitoring of pesticide
residues and even of toxic trace metals in the environmental
components, for the entire region and/or the whole country,
be established in that comparisons of residue data for
instance in a single fish species over a wide geographic
range will permit valid judgements of the regional diffe-

rences in pollution levels.

APPENDIX

38

APPENDIX

Table 6 - List of pesticides in use and/or in stock at the
International Institute of Tropical Agriculture

(IITA)

 

Actellic 25
Actellic Dust
Agrothion
Aldrex T
Aldrin Dust
Ambush ULV
Baygon 20
Baygon Aerosol
Cymbush Super
Decis/Dimethoate
Decis EC

Decis ULV
Detia

Didimac
Difolatan
Dimecron
Durban 4

Furdan 56

Pirimiphosmethyl
Pirimiphosmethyl
Fenitrothion

Aldrin + Thian
Aldrin

Permethrin

Propoxur

Propoxur
Cypermethrin

Decis + Dimetheoate
Decis + Enosulfan
Decis + Demitheoate
Alum Phosphide

DDT

Captafol
Phosphamidon
Chlorpyrifos

Carbofuran

25 D

5 ULV

ED
ULV
EC
ULV
57%
25 EC
80 WP
50 SWC
40 EC

56

39

Table g - (continued)

Trade Name
ESEQQ'ISE """"
Furdan 35 8+
Gammalin 20
Gammalin Dust

Lindane Dust

Kelthane

Malathion
Navan

Nogeos
Nuvacron EC
Nuvacron ULV
Orthene 75
Phostoxin Tabs
Pirimor ULV
Roach Pruff

Roqar/
Perfekthion

Sevin 85

Sherpa Plus EC
Sherpa Plus ULV
Thiodan 25 ULV
Thionex
Ultracide

Vetox 5

Common Name

Lindane
Lindane
Lindane

Chlorophenyl Trio-
chloroethanol

Malathion
Dichloroous

DDVP

Monocrotophos
Monocrotophos
Acephate

Aluminum Phosphate
Pirimicarb

Boric Acid

Dimetheoate
Carbaryl

Cypermethrine

Endosulfan
Endosulfan
Methidathion - ?

Carbaryl

Active Ingredient

50 EC
100 SC
50 EC
40 EC
ULV

75 WP

40 EC

85 WP
EC
ULV

25 ULV
EC

40 EC

40

Table g - (continued)

Trade Name

Vetox 85

Vetox 85

COIIIIROI'I Name

Cabaryl

HERBICIDES

Active Ingredient

Aatrex
Amiben
Atranex

Avirosan

Blazer
Cobex
Cotoran

Cotoran Multi

Dachtal
Dual
Enide

Galex

Gesaprin
Gesatop
Hyvar X
Hyvar X
Hyvar XL

Karmex

Atrazine
Chloramben
Atrazine

Piperophos +
Dimethane

Acifluorfen
Dinitramine
Fluometuron

Fluometuron +
Metolachlor

DCPA
Metolachlor
Difenamid

Metolachlor +
Metobromuron

Atrazine
Simazine

Bromacil

Diuron

40

240

80

500

20

20

500

75

75

500

50

500

500

500

80

80

20

80

EC

EC

EC

EC

EC

EC

EC

WP

WP

EC

WP

FW

FW

FW

WP

WP

EC

WP

41

Table g - (continued)

Trade Name

Paraquat
Patoran
Patoran
Preforan

Primagram

Primextra

Prowl

Rinsane

Round Up
Ronstar
Sencor
Stam F—34
Stomp

Tamariz

Treflan
Weed Killer 66
Weedone LV-4

Wet ALD

Common Name

Alachlor
Gramoxone
Metobromuron
n
Fluorodifen

Atrazine +
Metolachlor

Atrazine +
Metolachlor

Pedimenthalin

Fluorodifen +
Proponil

Glyphosate
Oxadiazon
Metribuzin
Proponil
Pedimenthalin

Proponil +
Bethocarb

Trifluvalin
2,4-D Amine
2,4-D

Surfactant

Active Ingredient

50

670

30

500

500

420

300

36

25

70

360

330

200

480

WP

EC

FW

FW

EC

EC

WSP

EC

WP

EC

EC

EC

EC

60EC

60

EC

42

Table 6 - (continued)

FUNGICIDES

Benlate
Demosan
Difolatan
Fernasan

Maneb

Benomyl
Cholroneb
Captafol
Thiram/Lindane

Dithane

SOIL STERLIENTS

50 WP

65 WP

80 WP

25 D

46 WP

Basmid Granular
Telone II

Vapan

Dazomet
Dichloropropene

Metam-Sodium

FERTILIZERS

Ammonium
Sulphate

Calcium
Ammonium
Nitrate

Compound
NPK 15~15-15

Compound
NPK 26-12-0

Hydrate Lime

Iron Chelate

15-15-15

26-12-0
98% Ca(OH)2

138 FE

43

Table 9 — (continued)

Trade Name Common Name Active Ingredient
Magnesium

Sulfate 98% Ng SO4
Muriate of
Potash 60% K20
Single Super

Phosphate 18% P205
Triple Super

Phosphate 44% P205
Urea 46% N

Zinc Durham

Sulfate Mono-

hydrate 36% ZN
Boron Foliar Spray

Zinc Foliar Spray

Notes : EC Emulsible Concentrates
D = Dusts
ULV = Ultra-Low-Volume (Concentrates)
ED Emulsible Dusts
WP Wettable Powders
SWC = Sprayable Water Concentrates

SC = Sprayable Concentrates
DL = Dust
FW = Flowable

WSP = Water Soluble Powders

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44

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_,_,.-

 

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