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Histopathology in the fish Channa punctatus, Heteropneustes fossilis and

Anabas testudineus exposed to diazinon

Article · November 2019

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 International Journal of Fisheries and Aquatic Studies 2019; 7(6): 47-54

E-ISSN: 2347-5129
P-ISSN: 2394-0506
(ICV-Poland) Impact Value: 5.62 Histopathology in the fish Channa punctatus,
(GIF) Impact Factor: 0.549
IJFAS 2019; 7(6): 47-54 Heteropneustes fossilis and Anabas testudineus exposed
© 2019 IJFAS
www.fisheriesjournal.com to diazinon
Received: 21-09-2019
Accepted: 25-10-2019
Mohammad Sohidul Islam, MD. Mansurul Haque, MD. Nazim Uddin and
Mohammad Sohidul Islam
Department of Zoology,
MD. Hasanuzzaman
Jahangirnagar University,
Savar, Dhaka, Bangladesh Abstract
Pesticides deteriorate the normal function of vital organs of fishes and in case of high concentrations, it
MD. Mansurul Haque totally damaged those organs. Histological changes in gills, liver, heart, intestine and kidney of three
Department of Zoology, common fish species (Channa punctatus, Heteropneustes fossilis and Anabas testudineus) have been
Jahangirnagar University,
studies to observe the effect of Diazinon as it has become a very common pollutant in the aquatic
Savar, Dhaka, Bangladesh
environment. Six different concentrations (2.5. 5.0, 10.0, 15.0, 20.0 and 25.0 mg/l) of pesticide had been
MD. Nazim Uddin applied in the test aquaria (Chari) to experience the major alterations of the vital organs of the three
Directorate of Secondary and treated species of fish. Lower concentrations (2.5 to 10.0 mg/l) had no remarkable effect on the treated
Higher Education, Dhaka, organs, whereas, Curley gill lamellae, irregular blood vessels in the liver, muscle fiber destruction in
Bangladesh heart, blended Submucosa and glomerular necrosis were found in C. punctatus from 15.0 to 25.0 mg/l
doses. Lamellar fusion, sinusoid vacuoles, fused villi and hemorrhagic renal tubules were observed in H.
MD. Hasanuzzaman fossilis and bent in secondary lamellae, irregular blood vessels in liver, fragmented muscle fiber in heart,
Institute of Food and Radiation ruptured villi as well as necrotic glomerular were noticed in A. testudineus in the same doses of Diazinon.
Biology, Atomic Energy The present study demonstrated that pesticide (Diazinon) disrupted the normal function of the sensible
Research Establishment, organs of fishes by damaging them in different ways.
Bangladesh Atomic Energy
Commission, Ganakbari, Savar, Keywords: Histological alteration, pesticide, ecosystems, freshwater fishes
GPO Box- 3787, Dhaka- 1000,
Bangladesh
1. Introduction
Pesticides are regarded as important tools for pest control in agro-farm and forestry though it
has become the contributor to gradual aquatic ecosystem degradation that recommended as a
greater part of the natural environment [1, 2]. It boosts the crop yields in one hand, contaminates
the water bodies through spray drift and leaching from the soil surface that induces the
ecological balance and hazardous health effects on a diversity of non-target creatures including
fish on the other hand [3]. In recent years, Pesticides have become a growing concern of
bioaccumulation as well as the persistence of various pesticides in the aquatic environment
comprehended a great threat to the overall ecosystem including human beings [4]. Since 50
years ago pesticides present in surface waters were reported in Canada, North America and
Europe. After that a large number of reports had been documented revealing the toxic effects
of these pollutants to the aquatic ecosystem [5, 6]. C. punctatus, H. fossilis, A. testudineus,
Barbodes gonionotus and some other indigenous small fishes use paddy fields and adjacent
aquatic ground (canals, lakes, ponds, swamps and inland water bodies) as breeding and
nursing of their fries, hence they played as biological indicators of ecotoxicological studies.
Pesticides with high concentration attenuated the survival, growth and reproduction of these
indigenous fish species [7] and exposed some visible effects on fish. This is because, fishes are
very sensitive to contaminated water occurring by pesticides, and certain physiological and
biochemical processes may damage when pesticides enter into different organs of fish [8].
Organophosphates are extensively used pesticides in the agriculture sector and it accounts for
approximately 50% of global pesticide use (John, 2007). Diazinon (0,0-diethyl 0-[6-methyl-
Corresponding Author: 2(1-methylethyl)-4-pyrimidinyl] is a widely used organophosphate insecticide for agriculture
Mohammad Sohidul Islam and domestic pest control. It is also used to restrain a variety of Hymenopterans and
Department of Zoology, Hemipteran insects including aphids, beetles, scales and pill bugs [10, 11]. After application in
Jahangirnagar University,
Savar, Dhaka, Bangladesh
the agro-farm, Diazinon easily washes away into the surface water and ultimately brings into
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adjacent water bodies, ponds, rivers and lakes where they Jahangirnagar University, Savar, Dhaka, Bangladesh with the
contaminate the aquatic ecosystems and affects the aquatic photoperiod of 12D:12L. Fishes were fed twice daily with
organisms. The recent work that has been done on evaluating earthworm and dead fishes were immediately removed to
the effects of Diazinon on different species of fishes [12-23] and avoid possible water quality deterioration. The water of the
so on. Due to their low cumulative ability and short experimented Chari was changed regularly that maintained
persistence in the environment organophosphorus (Diazinon) the better possible effects of Diazinon.
insecticides have fully replaced the chlorinated insecticide in
1970’s and at the beginning of 1980’s [24]. Though 2.2 Insecticide formulation
organophosphorus pesticides are extensively used in fish Insecticide was purchased from the local authorized dealer in
culture in order to suppress some parasitic diseases i.e. Savar market. 200 ml stock solution was prepared following
monogeneoses and arthropodoses [25, 26], some important the EC% active ingredient (mg/l) with the formula of
organs i.e. kidney, gills, stomach, brain, muscles and genital (200×60)/1000= X (X= amount of Diazinon 60EC), 200-X=
organs are damaged by its residual effects. DW (distil water). Finally, 200 ml stock solution = (X+DW)
Fishes are the main source of protein in human food among ml. The desired dose concentrations for 20L tap water were
other aquatic organisms. So it is necessary to find out the formulated with the aphorism of S1V1= S2V2 (Here, S1=
adverse effects of pesticide pollutants on fish as they have a 200mg/l, S2= 1 mg/l, V1= dose concentration, V2 =20L). The
direct link with the food chain as well as contamination of selected concentrations of chemicals were poured into the 20L
water bodies occurring by pesticides imbalanced the aquatic tap water in the Chari with a micropipette and stirred the
system [27]. Fishes are severely affected due to insecticides in solution gently with a glass rod for mixing completely.
different ways, mostly affected to crucial organs i.e. gills,
liver, heart, kidney, intestine etc. Among them, gills are 2.3 Histological study (Dissection and Slide preparation)
primarily attracted because they are the first organs to be For the histological investigation, the fishes (20×3) were
exposed by pollutants [28]. The water-born toxic particles exposed to various concentrations of Diazinon (2.5. 5.0, 10.0,
infected kidney as it regulates the extracellular and 15.0, 20.0 and 25.0 mg/l) with three replications for each
composition of fluid volume as well as the acid-base balance group. The control group fishes (20) had not treated with
of fish. Insecticides disrupt the functions of the kidney and insecticide. After 04 days (96 hours), both the control and
most cases it causes temporary or permanently derangement insecticide treated fishes (randomly selected three fishes in
of homeostasis [5]. Through these backdrops, the present study each concentration) were dissected and examined for
has been conducted to investigate the toxicity effects of histological study by the use of dissecting tray and surgical
Diazinon on some histopathological indices of the three tools (scalpels, forceps, needle etc.) following the methods of
indigenous freshwater fish species i.e. C. punctatus, H. Keneko [29] and Schalm et al. [30]. Gill, Liver, Heart, Intestine
fossilis and A. testudineus that would facilitate knowledge for and Kidney were dissected sophisticatedly and kept in plastic
the management of freshwater reservoirs regarding Diazinon vial with 10% formaldehyde. After fixation for 24 hours, the
application in agriculture fields. sections of tissues were dehydrated by ethyl alcohol for
removed water fromthe tissue block and then embedded
2. Materials and Methods within a small cube of paraffin. The paraffin embedded tissue
2.1 Collection and maintenance of experimental fish blocks were sectioning accomplished by using a microtome.
species: Three experimented fish species- C. punctatus with The microtome drives a knife across the surface of the
12 to 16 cm length and 24 to 30 gm weight, H. fossilis with 13 paraffin cubes and produces a series of thinsections (5µm)
to 18 cm length and 25 to 32 gm weight and A. testudineus with acontinuous ‘ribbon’. Then the sections were mounted
with 4.3 to 7.1 cm length and 10.6 to 28.3 gm weight were on individual microscope slides and stained with hematoxylin
collected from the local water bodies (beels) in Dhamrai area andeosin. In the end, the sections of tissue were permanently
located in Savar district, Bangladesh. Selected fishes were mounted under a coverslip. After preparation, slides were
transported in plastic containers and reared in 5 clay pots viewed under Olympus CX41microscope (X10) and
locally called Chari of 18×5×10 inches in size with 20 liters photographs were taken for further analysis.
of water receptivity of each. All samples were cleaned with
0.5% KMnO4 solution for five minutes to set free external 3. Results
infections and acclimatized them under tap water in a large Histopathological observation of different concentrations of
glass tank with 100 liters capacity for a week. Water Diazinon was done on gills, liver, heart, intestine and kidney
temperature 26±0.17º C with pH 6.5±0.02 and 7.3±0.22 mg/l of three treated fish species are presented in Table 1. The
dissolved oxygen were maintained in the test Chari. The specific changes of the different organ of the insecticide
experiments were conducted in the Department of Zoology, exposed fishes are described herein-

Table 1: Alterations appeared in the selected organs of the experimented fishes due to insecticide (Diazinon) treated with different
concentrations
Name of Insecticide Effects on organs of fish
Fish species concentrations (mg/l) Gills Liver Heart Intestine Kidney
2.5     
5.0     
10.0     
Channa Curly and bent Blood vessels Necrosis and fragmentation Submucosa Hemorrhage renal
15.0
punctatus lamellae irregular muscle fibers disintegrated tubules
Rupture Submucosa Necrotic changes
20.0 Lamellar fusion Destruction blood vessels
sinusoids blended in glomerulus
25.0 Blending and Vacuoles in Fragmentation muscle fibers Villi fused/ Necrotic changes
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destructiongill sinusoids ruptured in glomerulus

arches
2.5     
5.0     
10.0     
 Destruction  Hemorrhage renal
15.0 Necrosis muscle fibers
Heteropneust blood vessels tubules
es fossilis Hemorrhage Serosa slightly Necrotic changes
20.0 Lamellar fusion Destruction blood vessels
blood vessels damaged in glomerulus
Destruction gill
Vacuoles in Villi fused Necrotic changes
25.0 filaments and gill Fragmentation muscle fibers
sinusoids in glomerulus
arches
2.5     
5.0     
10.0     
Curly and bent Blood vessels  Hemorrhage renal
15.0 Fragmentation muscle fibers
secondary lamellae irregular tubules
Anabas
testudineus Submucosa Changes in
Curly and bent Hemorrhage
20.0 Destruction blood vessels disintegrated and glomerulus
secondary lamellae central vessels.
vacuolated
Blending and
Vacuoles in Villi ruptured Necrotic changes
25.0 destruction Fragmentation muscle fibers
sinusoids in glomerulus
lamellae
 = No changes apparently

3.1. Channa punctatus 3.2 Heteropneustes fossilis

3.1.1 Gills: Fusion of secondary lamellae, epithelial lifting, 3.2.1 Gills: No changes of gills were found in lower
epithelial hyperplasia and degeneration of secondary lamellae concentrations upto 15.0 mg/l unit of insecticide treated fishes
were observed in the gills of fishes exposed to different for 96 hours. Lamellar fusion was first noticed in 20.0 mg/l
concentrations of Diazinon. Apparently, there were no concentration and in 25.0 mg/l level gill filaments and gill
changes of the desired organs were noticed in lower arches were found destructed condition (Fig. 1, E-G).
concentrations (2.5-10.0 mg/l) of the treated fishes.
Abnormalities were found with comparatively high 3.2.2 Liver: Histological observation of liver of both control
concentrations. Fusions of secondary lamellae were noticed in fishes and upto 10.0 mg/l concentrations of insecticide
15.0 to 20.0 mg/l concentrations. In 25.0 mg/l concentration exposed fishes in the present study showed a normal
level showed bending and destructions of gill arches (Fig. 1, homogenous mass of hepatocytes with no abnormalities. The
A-D). destruction of blood vessels was started from 15 mg/l dose.
Hemorrhage in blood vessels was found in 20.0 mg/l
3.1.2 Liver: Normal structures of liver tissues were found concentration level and Vacuoles were seen in sinusoids in
upto 15.0 mg/l concentrations of insecticide treated fishes. the highest concentration level (25.0 mg/l) in the present
Abnormalities started from 20.0 to 25.0 mg/l concentrations study (Fig. 2, D-F).
level. Sinusoids and bile duct of liver were ruptured in those
levels, irregular blood also found in the treated fishes (Fig. 2, 3.2.3 Heart: Treated fish’s heart tissues showed minor
A-C). changes at low concentrations 2.5 to 10.0 mg/l. In higher
concentrations from 15.0 to 25.0 mg/l, the tissue exhibited
3.1.3. Heart: Some significant changes were found after remarkable changes including necrosis, destruction of blood
treated with the 20.0 mg/l concentrations ofthe insecticide for vessels and fragmentation of muscle fibers of heart (Fig. 3, D-
96 hours exposure period in the fishes. In 25.0 mg/l the G).
sections of the tissue showed congestion of blood vessels and
fragmentations of muscle fibers (Fig. 3, A-C). 3.2.4 Intestine: Intestine of H. fossilis was not significantly
infected in lower concentrations of insecticide (2.5-15.0
3.1.4 Intestine: The intestine of C. punctatus has a mucosa, mg/l). From 20.0 to 25.0 mg/l unit of concentrations
submucosa, muscularis and serous membrane among them muscularis swollen, disintegrated sub-mucosa, slightly
serosa was the outermost protective cover of intestine. Lower damaged serosa and fused or ruptured villi were found at 96
concentrations level (2.5- 10.0 mg/l) had shown no changes in hours observation period (Fig. 4, D&E).
the membrane of intestine but from apparently higher
concentrations submucosa was disintegrated in 15.0 mg/l dose 3.2.5 Kidney: Kidney tubules and haematopoietic cells were
and in 20-25mg/l of concentrations blended mucosawith found normal both control and insecticide treated
rupture villi were found (Fig. 4, A-C).
concentration limits upto15.0 mg/l. From 20.0 to 25.0 mg/l
Diazinon 60 EC promoted necrosis of tubular and
3.1.5 Kidney: Kidney tubules and haematopoietic cells were
haematopoietic cells of the kidney. Hemorrhage of renal
normal forms the concentration limits of 10.0 mg/l. From 15.0
to 25.0 mg/l level, degeneration of renal tubules, Bowman’s tubules and necrotic changes of glomerulus were also
capsule with atrophying glomeruli and severe hemorrhage observed in these concentrations level (Fig. 5, D&E).
were observed (Fig. 5, A-C) in the insecticide treated fishes.

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3.3 Anabas testudineus were attained in ruptured and fused condition (Fig. 4, F&G).
3.3.1 Gills: Lower concentrations of Diazinon had not
remarkably affected to gills of A. testudineus and it has gone 3.3.5 Kidney: Kidneys of the experimented fishes became
upto 10.0 mg/l. Curly form and bent of secondary lamellae infected by Diazinon at 15.0 mg/l concentration. At that stage,
were noticed from 15.0 to 25.0 mg/l concentrations. Lastly, renal tubules were hemorrhagic while glomeruli were
the gill lamellae were found destructed in the present highest changed in 20.0 mg/l concentration level. Severe necrosis in
treated dose (25.0 mg/l) (Fig. 1, H-J). tissues was observed in 25.0 mg/l dose at 96 hours of the
3.3.2 Liver: Liver tissues were normal upto 10.0 mg/l observation period (Fig. 5, F&G).
concentrations of both control and insecticide treated fishes.
Abnormal structure of blood vessels and sinusoids started
from 15.0 to 25.0 mg/l concentrations level. Irregular blood
vessels were found in 15.0 mg/l concentration unit and it turns
to hemorrhagic in 20.0 mg/l level. Severe necrosis in liver
tissues was observed in this concentration. Some vacuoles
were seen in sinusoids in the highest exposed concentration
(25.0 mg/l) of insecticide at 96 hours observation period (Fig.
2, G-I).

3.3.3 Heart: Apparently no changes were found in lower

concentrations (2.5 to 10.0 mg/l) of Diazinon treated fishes
compare with the control fishes while two major changes
were noticed in the higher concentrations (15.0 to 25.0 mg/l)
of insecticide treated fishes. In these concentrations level
fragmented muscle fibers and destructed blood vessels were
found in the heart tissues of the experimented fish samples
(Fig. 3, H-J). Fig 1: Histological photomicrograph of control and diazinon affected
gill tissues of three fish species; A-D = C. punctatus; E-G = H.
fossilis; H-J = A. testudineus; * = control, + = 20 mg/l; ++ = 25 mg/l
3.3.4 Intestine: Diazinon concentrations upto 15.0 mg/l had
concentration; GL= gill lamellae; GA= gill arch; TB= test buds;
not affected to the intestine of A. testudineus alike to the FSL= fusion of secondary lamellae; BGA= bend of gill arch; DGA=
control group samples. In 20.0 mg/l concentration, the damage of gill arch; GF= gill filament; DGF= destruction of gill
submucosa layer was found disintegrated and vacuolated and filament; CSL= curly of secondary lamellae; DGL= destruction of
in the highest treated dose of 25.0 mg/l level intestinal villi gill lamellae.

Fig 2: Histological photomicrograph of control and diazinon affected liver tissues of three fish species; A-C = C. punctatus; D-F = H. fossilis; G-I = A.
testudineus; * = control; + = 20 mg/l; ++ = 25 mg/l concentration; S= sinusoids; BV= blood vessel; RS= rupture of sinusoids; BD= bile duct; DBV=
destruction of bile duct; H= hemorrhage; V= vacuole; IRBV= irregular blood vessel; N= necrosis.

Fig 3: Histological photomicrograph of control and diazinon affected heart tissues of three fish species; A-C = C. punctatus; D-G = H. fossilis; H-J = A.
testudineus; * = control; + = 20 mg/l; ++ = 25 mg/l concentration; MT= muscle tissue; BV= blood vessel; MF= muscle fiber fragmentation.
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rapid changes of tissues depend on the concentrations of

insecticides and the time duration that fishes are exposed to
the toxicants [32]. Some histopathological studies evidences
such as, liver tissue lesions were found in freshwater fish
Cirrhinus mrigala [33], common carp Cyprinus carpio [34] after
10 and 30 days treated to sub-lethal concentrations of
Dichlorvos and Diazinon respectively. Histopathological
alterations of different tissues in Diazinon treated fishes have
also observed from the study of Deltamethrin treated fishes by
Cengiz [35], Cengiz and Unlu [36], Fenitrothion treated studies
by Benli and Ozkul [37, 38]. The lesions of different
sophisticated organs i.e. gills, liver, heart, kidney and
digestive truck of various insecticides treated fishes disturbed
homeostasis that leads to physiological disorders and
consequently dead of these fishes. The results of the present
study revealed that Diazinon severely affected the gills of the
three treated (C. punctatus, H. fossilis and A. testudineus) fish
species where curl and bent of secondary lamellae, lamellar
fusion and gill filaments and gill arches were destructed in
different concentrations. Similar results reported from other
studies. Mallatt [39] and McKim and Erickson [40] showed that
gills were the main target tissue induced by Diazinon that
Fig 4: Histological photomicrograph of control and diazinon affected used as the main route of arrival pesticides. In fish, gills are
intestine tissues of three fish species; A-C = C. punctatus; D&E = H. regarded as the vital organ, work as their respiratory
fossilis; F&G = A. testudineus; * = control; + = 20 mg/l; ++ = 25 osmoregulatory and secretory functions. Irregular and
mg/l concentration; M= muscular tissue; S= sub mucosa; V= villi; decrease of respiration are the early symptoms of insecticide
MSW= muscularies swollen; RV= rupture of villi toxicity [35] that dominate the physiological functions and may
cause the death of the fish. Khosrava-Katuli et al. [41] studied
the Diazinon effects on Caspian roach Rutilus rutilus. After
96 hours of exposure, they found shortening of secondary
lamellae, oedema, destruction of epithelial lamella, epithelial
lifting, curling of secondary lamellae, epithelial hyperplasia
and lamellar fusion. It fully supported the result of the present
study. Some other studies also noticed the similar effects of
insecticides on gills of different species of fish. For example,
histopathological effects of Deltamethrin on gills of Nile
tilapia Oreochromis niloticus were studied by Yeldrim et al.
[42]
, Atrazine induced of degenerative effects in the gill
epithelium of Gnathonemus petersii by Alazemi et al. [43],
Lambda-cyhalothrin effects on gills of Cirrhinus mrigala by
Velmurugan et al. [44]. Dutta et al. [45] showed that due to
Diazinon several changes occurred in Atlantic salmon
Salmosalar. Epithelial layer lifting, hyperplasia and necrosis,
shortening of the lamellae, frequent epithelial rupture,
lamellar fusion, mucous cells hypertrophy, extensive fusion
and clavate lamellae were found in the experimented fishes.
Oruc and Usta [46] reported that when Diazinon exposed to
common carp C. carpio lamellar fusion and epithelial
membrane lifting of gills are being observed supporting the
findings of the present study.
Histopathological analysis of the three species of Diazinon
Fig 5: Histological photomicrograph of control and diazinon affected treated fish samples revealed of the liver tissue alterations,
kidney tissues of three fish species; A-C = C. punctatus; D&E = H. including irregular and destruction of blood vessels,
fossilis; F&G = A. testudineus; * = control; + = 20 mg/l; ++ = 25
hemorrhagic in the blood vessel and rapture and vacuoles in
mg/l concentration; BC= bauman’s capsule; RT= renal tubule; BV=
blood vessel; G= glomerulus; H= hemorrhage; N= necrosis. sinusoids in the present study. These results are incorporated
into Cattaneo et al. [47] who reported that disorder in
4. Discussion hepatocyte’s cods, rupture in cell membrane, vacuoles in
Histopathological observation of different tissues of treated sinusoids of the liver tissues of silver catfish, Rhamdi aquelen
fish species is work as a catalyst for toxicological studies and for the effect of 2,4-dichlorophenoxiacetic acid. It also
monitoring of water quality of the aquatic ecosystem. followed the results of Cengiz and Unlu [36], Mishra and
Structural changes of tissues in the experimented fishes Mohanty [48] and Vinodhini and Narayanan [49]. They reported
exposed to different concentrations of insecticides play a that hypertrophy of hepatocytes, circulatory disturbances,
significance role as of active response to the organisms that focal necrosis, narrowing of sinusoids were seen in Gambusia
facilitate knowledge of the nature of toxicants [31]. Major and affinis and C. punctatus and C. carpio exposed to

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deltamethrin and heavy metal. The present study also 5. Conclusions

followed the finding of Matos et al. [50] and Sepici-Dincel et The results of the study concluded that even a small amount
al. [51]. They notice the similar histopathological changes in of pesticide (Diazinon) presence in fresh water reservoirs
liver tissues of O. niloticus and C. carpio exposed to sub- causes harmful effects on fish physiology and subsequently
lethal concentrations of carbaryl and cyfluthrinto the make death of the fish species. So, from the pesticides
experimented fishes. standpoint, it should take necessary precautions to applied
Different concentrations of Diazinon severely affected the pesticides in natural environment or use of pesticide in
kidney of the three species of treated fish samples. minimum quantity to protect the aquatic creatures. Therefore,
Hemorrhage in renal tubules, glomerular changes and necrosis further investigations required to select the optimum
in glomerulus were observed in the treated fishes. Similar concentrations of pesticides as well as histopathological
results were found from the studies of Glover et al. [52]. They changes in other organs of the experimented fish species.
exposed endosulfan on Atlantic salmon (Salmo salar) at a
concentration range from 4 to 710 µg kg-1 for 35 days 6. Acknowledgement
duration, where they found irregular structure and This research work is a part of first author’s PhD thesis.
malfunction of the kidney from low concentration to high
degree of insecticide formulation. Banaee [31] reviewed in fish 7. Conflict of Interest
exposed to 0.1 mg/l diazinon and noticed that glomerular There is no conflict of interest between authors.
disorientation, urinary tubular dilation and cloudy swelling of
kidney tissues while in case of 0.2 mg/l concentration level 8. References
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carbaryls on juvenile rainbow trout, Oncorhynchus mykiss. 29.
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