Effects of Butachlor on Density, Volume and Number of Abnormal Sperms in Caspian Kutum (Rutilus frisii kutum, Kamenskii 1901)
Morphological assessment of sexually mature Rutilus
frisii kutum Kamenskii 1901 caught from the rivers (Shirud, Khoshkrud,
Sepidrud and Chelavand Rivers) flowing in the southwest Caspian Sea region
was conducted and sperm volume, total sperm count and sperm concentration
of abnormal sperms were determined after exposing the spawners to 60%
herbicide butachlor (machete). Spawners under study were maintained in
tanks (1000 L) at the Shahid Ansari Teleost Fish Hatchery and exposed
to two different concentrations (25 and 75% of its LC50 value)
of butachlor. Results obtained indicate that exposure to high butachlor
toxicity (75% of its LC50 value) decreased sperm volume to
0.61 ± 0.42 cc in 2-3 year old fishes and to 0.55 ± 0.42
cc in fishes above 3 years of age, while that in fish exposed to low butachlor
toxicity (25% of its LC50 value) decreased to 1.55 ±
0.42 cc in 2-3 year old fishes and to 1.28 ± 0.42 cc in fishes
above 3 years of age. The sperm volume under normal conditions in R.
frisii kutum is 4.6 ± 0.42 cc in 2-3 years old and 4.58 ±
0.42 cc in fishes above 3 years of age. The total sperm count in R.
frisii kutum is 39.74 ± 2.5 billion spermatozoa cc-1
in 2-3 year olds and 42.99 ± 2.5 billion spermatozoa cc-1
in fishes above 3 years of age. When exposed to high butachlor toxicity,
total sperm count dropped to 16.92 ± 2.5 billion spermatozoa cc-1
in 2-3 year olds and to 15.98 ± 2.5 billion spermatozoa cc-1
in fishes above 3 years of age. Similarly total sperm count in R.
frisii kutum exposed to low butachlor toxicity was recorded as 23.6
± 2.5 billion spermatozoa cc-1 in 2-3 year olds and
29.4 ± 2.5 billion spermatozoa cc-1 in fishes above
3 years of age. Under normal conditions, on the basis of morphology, spermatozoa
showed only 10 ± 1.92% of abnormal sperms. The number of abnormal
sperms increased by 28.6 ± 1.92% in fishes exposed to high butachlor
toxicity, while that in fishes exposed to low butachlor toxicity increased
by 19.7 ± 1.92% in 2-3 year olds and 16.6 ± 19.2% in fishes
above 3 years of age. It is evident from the results obtained that increase
in level of pollution caused a decrease in sperm volume but an increase
in the percentage of abnormal sperms.
Rutilus frissi kutum is the most important of teleosts inhabiting
the Caspian Sea which comprises the maximum percentage of annual catch
for bony fishes in the Caspian Sea (Nikou et al., 2007). This species
is an anadromous cyprinid which enters rivers in the south Caspian Sea
for spawning (Bahmani et al., 2007). During its spawning migration
from the sea to the river, it remains in the estuary until it regulates
the internal water and total solute concentrations of its body fluids
to that of the freshwater (Bahmani et al., 2007). Spawners are
caught from rivers in the southwest Caspian Sea region, 30-300 m from
the river estuaries for semi artificial breeding programs. Spawners enter
the rivers to attain complete sexual maturity of gonads in freshwater
and are ready for spawning when they reach the spawning ground. Predicting
the effects of environmental hazards on spermatozoa alterations can be
useful in developing new technologies for the artificial breeding of this
commercially valuable species of the Caspian Sea (Alavi et al.,
It has been demonstrated that sperm motility, density, viability and
quality is significantly affected by salinity, osmotic pressure, ionic
composition and pollution (Bahmani et al., 2007; Carter, 2006;
Alavi et al., 2004). The effects of pollution on sperm activity
have also been studied in Cyprinis carpio and Acipenser persicus
(Alavi, 2004). Butachlor is one of the most popular herbicides used to
control weeds in transplanted rice paddy fields. With regard to the fact
that most paddy fields are located on the banks of rivers, the run-off
contaminated by this pesticide may affect the reproduction and physiological
characteristics in fishes during their spawning migration (Esmaily Sari,
2002). Exposure to pollutants of any intensity may cause stress in aquatic
organisms. Stress is the collective physiological response of an individual
to maintain hemostasis (Pickering et al., 1982; Chen and Sontegard,
1984; Montpetit and Steve, 1998). The inhibiting effects of stress on
fish reproduction cause adverse effects on the functioning of endocrine
glands which result in decreased gamete and larval quality (Contreras-Sanchez,
1998). Apart from corticosteroids, sex steroids have also been recognized
as primary messengers of stress response in fishes (Scherek et al.,
2001). Stress is also a potent inhibitor of hormones such as gonadotropin,
testosterone, 11 keto-testosterone and 17-β estradiol in fish (Kubukawa
et al., 1999; Bayunova et al., 2002). Butachlor, N-butoxymethyl-2-chloro-2`
6`-diethyl acetanilide (Esmaily Sari, 2002) is the most commonly used
herbicide, the biochemical composition of which is very close to estrogen
(Esmaily, 2002). It is orange to brown in color with a molecular weight
of 311.9 and a specific weight of 1.07 at 25 °C. It has a melting
point of 156 °C and freezing point of < -5 °C. Butachlor has
a vapor pressure of 4.5x10-6 mm Hg at 25 °C and decomposes
at 165 °C (Piri Zirkoohi and Ordog, 1997).
Butachlor is used for the control of weeds particularly in paddy fields.
It is a selective herbicide that acts on soil and controls the growth
of roots and also prevents emergence of weeds. It is particularly effective
in controlling broad leaf arrowhead, Sagittaria pigmaea and the
barnyard grass Echinocloa crus and Alisma canaliculatum.
Butachlor is not very soluble in water and is sometimes mixed with propanil
to control unwanted weeds. Exposure to hormones or biochemicals which
are opposite to their sex hormones results in changes or termination of
sexual axis (McCormick, 2007). Thus butachlor mimics estrogen and decreases
testosterone levels in blood plasma. Growth and development of testes
and development of sertoli cells affect the number, density and volume
of spermatozoa (Schultz et al., 2003).
The effects of pollutants occurring in nature which are similar to estrogen
in composition have been studied which include effects of natural estrogen
in Zebra fish, Danio rerio (Anderson et al., 2003) and Rutilus
rutilus (Liney et al., 2005). Similar studies have also been
conducted on rainbow trout (Schultz et al., 2003). The effects
of increasing osmolarity as a result of pollution on the number of spermatozoids
were studied in Rutilus frisii kutum (Bahmani et al., 2007).
The minimum and maximum number of sperms in Rutilus frisii kutum
are 32 billion and 48 billion sperms cc-1, respectively (mean
= 40 sperms cc-1) and the sperm volume in kutum ranges from
a minimum of 2 cc to a maximum of 6.4 cc (Azari Takami and Razavi Sayed,
1990). Malformed sperms with double heads, sperms bound to each other
by their heads, or the head bound to the tail of other spermatozoa have
been reported in kutum resulting from inappropriate maintenance of spawners,
cold and/or hot shocks, toxicity and pollution and changes in acidity
(Vladi et al., 2002). Spermatozoa with 10% anomalies have been
reported in kutum (Bahmani, 2007). Based on Kortet et al. (2004)
this is considered high sperm quality.
In the present study, fish under study were exposed to the herbicide
butachlor in the hatchery and the effects of the herbicide were evaluated
on spermatogenesis in terms of sperm volume, number of spermatozoids and
number of abnormal spermatozoids.
With regard to the fact that chlorinated herbicides are capable of accumulating
in steroid tissues (Esmaily Sari, 2002) the present study was conducted
on two age classes to evaluate the changes in indices studied and thus
offer recommendations for enhancement measures in the aquatic environment
and reduction in the use of pollutants.
Considering the significance of Rutilus frisii kutum in the economy
of the coastal dwellers and the need for suitable male spawners possessing
good quality sperms in artificial breeding programs, the present investigation
was carried out on this species to determine the physiological alterations
in sperms caused by the extensive use of these herbicides in the study
region, particularly during the spawning migration of the target species.
These alterations in spermatozoa can be used as a model to predict environmental
hazards and thus assist in developing suitable measures to protect these
MATERIALS AND METHODS
Specimens and Experimental Set up
This study was conducted at the Shahid Ansari Hatchery from February
2007 through April 2007. Male spawners required for the study were randomly
collected from the Sepidrud, Khoshkrud, Khaleh Sara and other rivers located
in the southwest region of the Caspian Sea. Two doses, high dose (75%
of LC50 = 0.3225 ppm ) and low dose (25% of LC50
= 0.1075 ppm), were used for the herbicide butachlor that were based on
the LC50 values for this herbicide (APHA, 1986; Simmons, 1993).
The LC50 for butachlor for Rutilus frisii kutum was
determined as 0.43 ppm which correlated with the previous findings of
Piri Zirkohi et al. (2000). To study the effect of age, fishes
under study were divided into two age classes-the first group with fishes
belonging to the 2-3 year age class and the second group with fishes above
3 years age class. A control group was also studied simultaneously. The
split plot design was used with three replicates for statistical analysis
of results. The herbicide doses were applied one each per whole plot and
different age classes were applied on each subplot. Fiber glass tanks
(1000 L) equipped with aeration were used in a closed system at the Shahid
Ansari Hatchery in Rasht. A total of 540 kutum fish caught from the rivers
in the Gilan Province were stocked in these tanks. Age determination of
fish was carried out using scale method. After stocking fish were exposed
to the herbicide for six days. The toxic effects of the herbicide are
neutralized within 6-10 days in clean water (Esmaily Sari, 2002).
Daily Experiments and Sampling
Dissolved oxygen concentrations, pH and water temperature were measured
using the portable Multimeter and recorded. Fish are maintained in clean
water for 6 days to neutralize the effects of 6-day exposure to herbicides
and random samples are collected to measure total length (cm), total body
weight (g) and weight of gonads in spawners prior to sperm collection.
Sperm Collection and Spermatological Experiments in vitro
Sperm were collected by applying gentle abdominal pressure to
extrude the milt. Care was taken to avoid contamination with seawater
or urine (Alavi et al., 2004). The milt was transferred to a glass
vial and kept at 1-2 °C until its use. The concentration of spermatozoa
was determined using the haemocytometer method. In this procedure a 1:600
dilution (sperm:water) from each well-mixed sample was prepared (Kortet
et al., 2004) and semen volume was determined using a wide mouthed
pipette (Bahmani, 2007). The staining technique was used to differentiate
abnormal spermatozoa using a light microscope (Sherek et al., 2001).
The results obtained were analyzed using one way ANOVA and comparison
of means was carried out using SPSS (Vladi et al., 2002).
Sperm volume and sperm count as well as percentage of abnormal spermatozoa
were significantly affected by the dose of butachlor used at a 1% significance
level. Sperm density and percentage of abnormal spermatozoa also showed
significant correlations with total length and body weight at a 1% significance
level. Although no correlations were detected between age and sperm volume,
significant interactions were observed with different concentrations of
the herbicide butachlor on age, total length, sperm density and percentage
of abnormal spermatozoa at a 1% significance level (Table
Comparison of means of results obtained using Duncan`s Multiple Range
Test (MRT) at a 5% significance level indicates that exposure to butachlor
decreased sperm volume whereby lowest sperm volume (0.55 mL) was recorded
in the high dose treatment. Sperm volume did not change significantly
with age and decrease in sperm volume in both age classes studied was
not significantly different from a single control (Table
Comparison of means of sperm count and sperm density using Duncan`s test
indicate that high toxicity caused by the herbicide butachlor caused severe
declines in sperm count in Rutilus frissi kutum resulting in 16.37
billion spermatozoa. Butachlor toxicity in 2 year old fish was greater
and sperm count in this group declined to 26.7 billion spermatozoa. Exposure
to butachlor also resulted in abnormal spermatozoa and highest percentages
of these spermatozoa (28.6%) were recorded fish exposed to high dose of
butachlor, while percentage of abnormal spermatozoa in 2 year-old fish
was recorded as 17.98%.
|| Mean-squares of characteristics and doses of herbicide
butachlor in different age groups
|**Significant at 1 and 5% significance level
|| Comparison of means of parameters in male Rutilus
frissi kutum using Duncan`s MRT at 5% significance level
|Mean values with the different letter(s) significant
different at 5% significant level
|| Correlation coefficients detected between parameters
studied in Rutilus frissi kutum
|**Significant at 5% level
|| Variations in total length and sperm count as a function
of different doses of butachlor
|| Variations in total length and sperm volume as a function
of different doses of butachlor
Comparisons of correlations between parameters studied indicate linear
correlations between total length and total weight at a 1% significance
level. No correlations were found between sperm volume and percentage
of abnormal spermatozoa, however sperm volume showed linear correlation
with sperm count at 1% significance level and negative correlation with
percentage of abnormal spermatozoa at 1% significance level. Negative
correlations were also found to exist between sperm count and percentage
of abnormal spermatozoa (Table 3).
Increase in butachlor concentration severely decreased sperm count (Fig.
1). Increase in butachlor concentrations also resulted in decrease
in sperm volume (Fig. 2). Regression analysis using
dependent and independent variables were also carried (Fig.
3). Step by step analysis indicate that the parameters studied showed
strongest correlations with abnormal spermatozoa and last of all with
Understanding the ecology of different species of fishes in an aquatic
ecosystem is an important factor in the conservation and rehabilitation
of their stocks. Improving natural spawning areas and natural reproduction
can be effective in meeting the increasing needs of aquaculture in the
world (Moyle, 1991). Results obtained from morphological studies of spermatozoa
in Rutilus frissi kutum indicate that they a round to oval shaped
head with a cylindrical and elongated midpiece and long tail. This conformed
to the sperm morphology reported in carps, trouts and pikes (Krasznai
et al., 1995). Increased percentage in morphologically abnormal
spermatozoa strongly affect the fertilization rate in fishes and result
in the production of abnormal specimens. Careful handling and proper storage
are primary factors in maintaining semen fertility. Cold and/or heat shocks
and water pollution decrease stored sperm viability (Vladi et al.,
2002). Percentage of abnormal sperms in Rutilus frissi kutum was
reported as 10 % in the Shirroud, Tonekabon and Khoshkroud Rivers (Bahmani
et al., 2007). After exposure to the herbicide butachlor the percentage
of abnormal sperms increased to a maximum of 29.2% (mean = 20.6%) which
is a physiological response of fish to stress following exposure. Reduced
head, short or degenerated flagellum and/or flagellum coiled around the
head were among the various morphological changes observed in spermatozoa.
Kortet et al. (2002) regarded semen with 5-15% abnormal sperms
as good quality semen. Based on the results obtained from the present
study semen quality in fishes examined in this study is graded as average.
Semen quality in the two year old age class was poorer and this may be
related to the increased pollution uptake in testes in this age class.
Sperm volume is also influenced by species, number of samples taken, age,
weight, race and skill with which semen samples are taken. Azari Takami
and Razavi Sayed (1990) reported the minimum and maximum sperm volume
in Rutilus frissi kutum in the Hevigh River as 2 and 6.2 cc, respectively
(mean = 4.2 cc). In another investigation carried out in the Shiroud,
Tonekabon and Khoshkrud Rivers, mean sperm volume in this species was
reported as 3.87 cc (Bahmani et al., 2007). In the present study
mean sperm volume in Rutilus frissi kutum after exposure to herbicide
butachlor decreased to a minimum of 0.55 cc. Mean sperm volume in the
control group was recorded as 4.9 cc. The effect of age on semen volume
was not significant. Sperm count in Rutilus frissi kutum in the
Hevigh River ranged from a minimum of 32x10-6 to a maximum
of 48x10-6 spermatozoa (mean = 40x10-6 spermatozoa)
(Azari Takami, 1990), while that in specimens collected from the Shiroud,
Tonekabon and Khoshkrud Rivers was reported as 33.61x10-6 spermatozoa
(Bahmani, 2007). Toxicity caused by high dose of butachlor resulted in
severe decrease in sperm count (16.37x10-6 spermatozoa) in
this species. This decrease was more pronounced in the two year old age
class. Sperm count in the control group was reported as 41.3x10-6
spermatozoa. Most of the recent investigations on the effects of pollution
on reproduction aim at determining the maximum tolerable concentration
(MTC) of the contaminant because they are of the opinion that some of
the stages of life can be more sensitive (Esmaily Sari, 2002). Most of
these investigations are designed to determine the MTC for different fish
species during different stages of growth by focusing on the physiological
responses associated with exposure to pollution (Esmaily Sari, 2002).
Some pollutants directly affect sexual maturity and levels of sex hormones
such as testosterone and ultimately on spermatogenesis. The toxic effects
of cadmium have been documented in several shown to cause variations in
testosterone levels in blood plasma. This has been documented in minnows
(Phoxinnus phoxinus) as well as in carp and rainbow trout. Decline
in testosterone levels was obvious after 3-4 weeks exposure to PCBs .
It has been proved that the herbicide butachlor is similar in structure
to estrogen (Esmaily Sari, 2002) and its entry into aquatic ecosystems
can result in physiological changes in aquatic organisms. There are different
pathways for the entry of natural estrogen into the ecosystem. Some of
the major pathways through which estrogens can enter into the ecosystem
are industrial effluents from paper and paint production, industrial detergents
and agricultural effluents (Johnson et al., 2005). Of the estrogen
like compounds, 17 α-ethinyl estradiol (EE2) has received considerable
attention (Johnson et al., 2005). Estrogenic activity is strongly
associated with sludges produced during wastewater treatment (Anderson
et al., 2003). Estrogen and its analogue can strongly inhibit sperm
production. Similar results have also been documented in human (Groves
and Batten, 1986). Estrogen-like compounds have also been reported to
effect gonad tissue (Van Der Van et al., 2003) and have suggested
a possible link between estrogen-like compounds and reproduction indices
such as sperm count, gonad weight and gonadal indices in male fish (Nielson
and Baatrup, 2006). Inhibited development of testes and the formation
of sertoli cells may be related to exposure to estrogens (Dalgaard et
al., 2002). Decrease in production of sertoli cells decreases sperm
production as a result of estrogen activity (Atanassova, 2005). Stress
is one of the adverse effects of pollution in fish which alters hemostasis
(Jensen, 1983). Physiological change in the endocrine system is considered
a primary response to stress (Nikou et al., 2007). Nikou et
al. (2007) reported a significant decrease in testosterone levels
in Rutilus frissi kutum from 18.9 mg mL-1 at the time
of catch to 4.8 mg mL-1 one hour after catch subjected to stress
during handling and transport. Exposure to herbicide butachlor results
in estrogenic effect and also reduced testosterone levels in fish. All
fish have both testosterone and estrogen in their blood that are bound
by receptors which influence growth (Takahashi, 1975; Mayer, 2004), development
and behavior and regulate reproductive cycles and exert a positive feedback
on the reproduction axis (McCormick, 2007). But if these hormone levels
are perturbed, they exert a negative feedback on the hypothalamus and
inhibit the secretion of GnRH (Nagahama, 1983) and spermatogenesis of
sertoli cells and thus stop sperm production and cause variations in sperm
volume (Nielson and Baatrup, 2006). The effects of estrogen mimicking
compounds have been evaluated in rainbow trout (Shultz et al.,
2003). The results of the present study show that exposure to the herbicide
butachlor, which is an estrogen mimicking compound results in a decrease
in sperm volume and sperm count and an increase in the percentage of abnormal
spermatozoa. These findings are in line with results of Carter (2006)
which state that low toxicity influences reproduction and directly affects
free gametes such as sperms. Based on the theory of `sperm competition`
males occupying disfavoured mating roles compensate by having larger gonads
for their body size (Stockley et al., 1997) and by producing sperms
with increased sperm velocity, rather than longevity (Kortet et al.,
2004). The effects of herbicide butachlor in water goes even beyond this
because its estrogenic effects results in significant decreases in steroid
hormones particularly testosterone during stress (Bayunova, 2002). We
may therefore conclude that treatment with lower concentrations may produce
sublethal effects which are sometimes difficult to discern but, nevertheless,
important from a behavioral or ecological standpoint. Although chemicals
may be accumulated in the fish without causing their death, there are
many possible chronic effects on the living community. Careful evaluation
of chemical use cannot therefore be overemphasized. Efforts should be
taken to regulate pesticide use in order to prevent their entry into rivers
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