Effect of Metasystox-R on Marine Nitrosomonas sp. as a Nitrification Inhibitor
Metasystox-R is a systemic soluble liquid insecticide
for the control of aphids on brassica vegetable crops, cotton and lupins
and it is possible enter to the marine environment and may be have a hazard
effects for the marine organisms and nitrification processes. Effect of
Metasystox-R on ammonia oxidizing activity by marine Nitrosomonas
sp. was investigated by determining nitrification inhibitor assay in the
cell suspension. Results showed that 8 μg mL-1 of Metasystox-R
with PI50 = 4.48 significantly inhibited nitrite production
by marine Nitrosomonas sp. These results suggested marine Nitrosomonas
sp. may be one of the target bacteria which was inhibitor and decreasing
nitrification in the marine environment.
Key words: Metasystox-R, nitrification inhibitors,
marine Nitrosomonas sp.
Major transformation of soluble inorganic compounds occur
on fresh water and sea water. Remains of cell types of biota (detritus)
sink to bottom of the water body, decomposing to ammonia (Austin, 1989).
The process of nitrification is carried out by Nitrifying
bacteria (Hu et al., 2003; Murakami et al., 1995). Nitrosomonas
and Nitrobacter which was widely distributed in sea water and significance
in nitrogen cycle. The nitrification process is primarily accomplished
by two groups of autotrophic nitrifying bacteria that can build organic
molecules using energy obtained from inorganic sources, in this case ammonia
or nitrite (Kim et al., 1997; Bock et al., 1989; Lopez et
al., 2003). In the first step of nitrification, ammonia-oxidizing
bacteria such as Nitrosomonas sp. oxidize ammonia to nitrite according
to Eq. 1 (Watson et al., 1981).
We have already reported the nitrification inhibitory activity
of some compounds such as 2-chloro-6-(Tricholoromethyl) pyridine, 2-amino
4 methyl-6- trichloromethyl-1,3,5 triazine (MAST), on the nitrification
activity by Nitrosomonas europaea ATCC 25978 and Nitrosomonas
sp. TK794 (Ohki et al., 1999; Murakami et al., 1993;
Takagi et al., 1994).
Inhibitory activity of some compounds have been investigated
with intact nitrified bacteria cells and inhibitor compound, to conclude
that the inhibitors effect the ammonia oxidizing process to nitrite. In
these 15 years, more than 20 species of ammonia-oxidizing bacteria have
been isolated from waste water and sea water (Matsuba et al., 2003;
Mizoguchi et al., 1998) which was mostly belong to Nitrosospira,
Nitrosococcus and Nitrosolobus species.
Metasystox-R (ODM) is an organophosphorus compound. ODM
systemic insecticide for control of aphids on brassica vegetable crops,
cotton and lupins and establishment of maximum residue limits for oxydemeton-methyl
in brassica vegetable crops, cotton, lupins and animal commodities (Australian
Pesticides and Veterinary Medicines Authority, 1998). The ecotoxicological
of Metasystox-R indicates high to moderate toxicity to fresh water and
sea water fish and very high toxicity to aquatic invertebrates (Anderson,
1987). Otherwise enter Metasystox-R into water, hazard to fish and daphnia
and other aquatic invertebrates. We have investigated to obtain effects
of Metasystox -R as inhibitor on the marine Nitrosomonas sp. as
a compound inhibitory of nitrification.
MATERIALS AND METHODS
The experiments were carried out in the laboratory of (NTB)
faculty of marine science, Tehran, since 14 June 2007.
Chemicals: Metasystox-R (oxydemeton-methyl) (ODM) C6H15O4PS2
||Composition of the Nitrosomonas medium
|The pH was adjusted at 7.6
Metasystox-R, from Bayer AG, Company Germany were used as nitrification
inhibitor in this study.
Bacteria and their incubation: Marine strain of Nitrosomonas
sp. were used as ammonia-oxidizing bacteria which is supply from SERA
company which is used as a nitrifying bacteria in the sea water. This
strain was incubated at 29°C with shaking at 200 rpm in the dark in 100
mL Erlenmeyer flasks containing 30 mL of liquid media (Murakami et
al., 1995; Takagi et al., 1994) was cultured in the Nitrosomonas
Medium (Watson et al., 1981; Austin, 1989). The composition of
Nitrosomonas medium mention in the Table 1.
Nitrification inhibitory activity Preparation of cell suspension:
Cell suspension from Nitrosomonas sp. was cultured in the Nitrosomonas
medium (Table 1). Ten milliliters of the preculture
growth for 10 days were transferred into 500 mL Erlenmeyer flasks containing
100 mL of the new liquid media (Murakami et al., 1995; Takagi et
al., 1994). The flasks were shaken at 200 rpm in the dark at 29°C
for 10 days. About 300 mL of their culture media were centrifuged at 8600
x g for 15 min at 4°C. Then the bacteria pellet was washed twice with
sterilized 20 mM phosphate buffer (pH 8, P-buffer) by centrifugation at
7200 x g for 15 min at 4°C. The harvested wet cells resuspended with P-buffer
to 5 mL (Murakami et al., 1995).
Determination of nitrification inhibition assay in cell suspension:
For determination Nitrification inhibitory assay in cell suspension
substrate solution and different concentration of Metasystox-R (ODM) were
Substrate solution: Ammonia sulfate (100 µg nitrogen per milliliter
in P-buffer) was used to the substrate solution for the ammonia-oxidation
(Murakami et al., 1995).
Different concentration of Metasystox-R: Ten milligrams of Metasystox-R
were emulsified in water with a few drops of DMSO to make a 5 mL of inhibitor
Cell suspension: The cell suspension described in 3.1 was diluted
with P-buffer which were about 1x106 cells mL-1.
Nitrification inhibition assay in cell suspension: Into a 2 mL
test tube, 1 mL of each substrate solution, 1 mL of the diluted cell suspension
and 0.016, 0.008, 0.004 and 0.0004 mg mL-1 of Metasystox-R
solution was added and the test tube was allowed to stand in 37°C water
bath for 30 min to incubate. The nitrite concentration was determined
at the start of reaction (0 min) and after 30 min incubation (Murakami
et al., 1995) by means of OD value at 540 nm according to MOOPAM
(1999) method which was determined sea water nitrite.
Effects of DMSO without Metasystox-R was also tested as
control and also another control (P-buffer) was used diluted cell suspension
and substrate solution without Metasystox-R.
The inhibitory rate at each concentration was obtained from
the following equation (Murakami et al., 1995).
The molar concentration of inhibitor which shows 50% inhibition
against the nitrite production relative to the control was estimated (Takagi
et al., 1994).
The inhibitory introduced were expressed as PI50,
the molar concentration of the inhibitor, which shows 50% inhibition (molar
I50) against the nitrite formation by ammonia-oxidizing bacteria
relative to the control. The nitrification inhibition introduced are expressed
as PI50 which was the negative logarithm of the molar I50
(Matsuba et al., 2003; Murakami et al., 1995).
RESULTS AND DISCUSSION
Effect of Metasystox-R (ODM) on ammonia oxidizing activity
by marine Nitrosomonas sp. from SERA company was investigated by
determining nitrite amount in the cell suspension assay. Results showed
that maximum inhibitory rate (62.5%) which was induced with 0.016 mg mL-1
concentration of Metasystox-R and after diluted ODM had showed reduce
inhibitory rate activity (Table 2). The results mentioned
the ammonia oxidation
The effect of Metasystox-R
on ammonia oxidation in cell suspension of marine Nitrosomonas
a: OD value at
the start of reaction indicate nitrite amount before adding
inhibitory and substrate, b: OD
Value after 30 min incubation with inhibitor and substrate,
PI50 value: Negative logarithms of the molar I50 in
soil-water and cell suspension bacteria, marine Nitrosomonas sp.
was too sensitive to the Metasystox-R and prevent oxidation of ammonia
to nitrite in the culture medium with inhibitor activity (PI50
= 4.48) with 8 µg mL-1 significant inhibitory
as shown in Table 2.
Metasystox-R was dissolved in DMSO and concentrations of
DMSO and P-buffer did not inhibit the nitrite production nor the nitrite
oxidation (inhibitory rate 0.0 %).
Our laboratory results showed that Metasystox-R had inhibitory
activity on the marine Nitrosomonas sp. with PI50= 4.48
which is show the moderate inhibitory activity.
and nitrapyrin (2-chloro-6- trichloromethyl-pyridine) are inhibitors of
the ammonia-oxidation Nitrosomonas europaea ATCC 25978 with inhibitory
activity (pI50) of 5.75, 7.12, 5.66, respectively (Kasahara
et al., 2002). We known inhibitor with pI50<4.35 were
be week activity and exhibiting pI50>6.4 more sensitive to
Nitrosomonas sp. other experiment showed that Nitrosomonas europaea
sensitive to the three inhibitors (MAST, Br-MAST, nitrapyrin) and compare
this result with waste water and marine Nitrosomonas sp. was showed
that less sensitive to the three inhibitors (Matsuba et al., 2003;
Okano et al., 2004).
Comparing the PI50 value in this experiment indicate
that the nitrification inhibitory activity may be due to a direct action
to marine ammonia-oxidizing bacteria and marine Nitrosomonas sp.
with PI50 = 4.48 which was significant inhibitory activity
on marine Nitrosomonas sp. so results were observed that Metasystox-R
with moderate to high effects on marine Nitrosomonas sp. and could
be important effect on nitrogen cycle in the marine environment. The ecotoxicological
profile of ODM indicates high to moderate toxicity to birds, moderate
toxicity to mammals, moderate toxicity to fish and very high toxicity
to aquatic invertebrate (Australian Pesticides and Veterinary Medicines
Further investigation is under way to make clear the site
of action of Metasystox-R nitrification inhibitors including isolation
of microorganisms such as fresh water and marine Nitrosomonas and
Nitrosococcus in our laboratory.
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