J. Pestic. Journal Sci. 38(1), of Pesticide 10–26Science (2013) DOI: 10.1584/jpestics.D12-053
10 T. Katagi
Review
Aerobic microbial transformation of pesticides in surface water Toshiyuki Katagi* Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 4–2–1 Takatsukasa, Takarazuka, Hyogo 665–8555, Japan (Received September 10, 2012; Accepted December 11, 2012) Aerobic transformation by microbial organisms is a dissipation process of pesticides in surface water, but the corresponding information is much less available as compared with their microbial degradation in soil. Bacteria freely floating in water or associated with suspended particles are the key microorganisms degrading pesticides; their species and populations, however, depend on sites and seasons. The various factors related to pesticide properties, experimental conditions and characteristics of surface water are involved in the complex control of microbial processes. Bottom sediment and macrophytes with associated biofilms not only act as sink for pesticides but also provide the habitat for both bacteria and fungi that degrade pesticides. More understanding of each factor is necessary to utilize laboratory biodegradation data for the refined assessment of pesticide behavior in surface water. Keywords: microbial aerobic transformation, surface water, bacteria, fungi, biofilm.
Introduction Surface water in rivers, ponds and lakes is possibly contaminated with pesticides via various routes either from a point source such as sewage plants and sewer overflows or a diffuse one along a water course, such as surface runoff and soil erosion. The contamination from point sources is reported to be dominant in several European catchments and constitutes 20–80% of the pesticide load in rivers.1) Water analysis along two German streams showed that direct runoff from farmyards due to cleaning spray equipment significantly contributed to the total pesticide load of seven insecticides and fungicides, while outlet from sewage treatment plants was an important source for 13 herbicides.2) The authors also reported that the applied amount of pesticide to the field correlated with its load to the streams, but runoff from cultivated fields was a greater contributor for hydrophilic pesticides. Pesticides are partitioned to constituents of surface water such as biota, suspended particles, and bottom sediment and transported by a water flow with concomitant chemical and biological transformation, as illustrated in Fig. 1.1,3,4) Partition and chemical processes are individually evaluated by standard batch adsorption–desorption, hydrolysis, and photolysis studies. A small-scale laboratory water-sediment system with a typical * To whom correspondence should be addressed. E-mail:
[email protected] Published online February 14, 2013 © Pesticide Science Society of Japan
sediment-water ratio of 1 : 3–1 : 10 (w/v) is utilized to evaluate biological processes in the assessment of pesticide behavior4); however, aerobic transformation and mineralization by microbes in surface water have been recently proposed as an additional study.5–7) The species and density of microbes are generally site-/season-specific and are significantly affected by the status of the water body. The seasonal stratification in lakes along with temperature changes controls the depth-dependent concentrations of oxygen and nutrients, and the change in the flow rate in rivers leads to a different concentration of particulates.8) Sediment-water interfaces showing various physical, chemical and biological gradients are deeply involved in the recycling of elements.9) Furthermore, sunlight with wavelengths shorter than 400 nm is reported to play a significant role in the production of dissolved organic carbons (DOC) from estuary sediments.10) In this review, we first discuss the relative contribution of each environmental process to the dissipation of pesticide in surface water together with the importance of the microbial process especially by bacteria and fungi. After introduction of the study design assessing aerobic transformation with its kinetic analysis, the aerobic transformation profiles of pesticides are examined through a literature survey by considering the involvement of specific microbes in surface water. The factors controlling microbial transformation in surface water are discussed. Finally, an overview summary is provided, including issues that remain to be solved. The chemical structure of each pesticide appearing in this review is listed in the Appendix.
Vol. 38, No. 1, 10–26 (2013)
Aerobic microbial transformation of pesticides in surface water 11
Fig. 1. Behavior of pesticides in surface water.
Processes Controlling Pesticide Behavior Pesticides undergo various processes, as illustrated in Fig. 1. In rivers and streams, the pesticide concentration at an entrance point is highly diluted by the water flow by a factor of >10,11) while dispersion predominates in dilution for a stagnant water body such as ponds and lakes, which have a much longer hydraulic residence time.12,13) The dilution factor is also estimated by hydrodynamic models14) or monitoring the concentration of a tracer (Cl−) in a water system.15,16) Depending on its physicochemical properties, pesticide is partly adsorbed or associated with suspended particles, colloids, and dissolved organic matters (DOM),4,17,18) reaches bottom sediment via sedimentation or diffusion,17,19) and escapes from the water body via volatilization.4,14) The sorption of pesticides is evaluated by an adsorption coefficient determined in a batch study, generally assuming a linear or Freundlich isotherm. Sedimentation loss is estimated for a fraction of suspended particulates adsorbing pesticides by considering an average depth of water and a settling rate.14,20) The two-layer model is conveniently used to describe the volatilization of pesticides from water to air.14,20) Abiotic hydrolysis follows the pseudo-first-order kinetics at a very dilute concentration of pesticide, and its contribution is usually limited at neutral pH and low temperatures except for pesticides having an ester linkage.17) In a clear water column, direct photolysis by sunlight plays a role in the dissipation of pesticides having a measurable U.V. absorption at >290 nm. A direct photolysis rate with seasonal and depth dependence can be theoretically estimated using a quantum yield of pesticide.21) Furthermore, indirect photolysis including reactions with active oxygen species such as singlet oxygen (1O2) and hydroxyl radical (·OH) becomes important for some pesticides.22) Its contribution is conveniently assessed by the typical reaction rate constants and steady-state concentrations of active oxygen species in natural water.14,20)
Microbial degradation is another important dissipation route, as is bio-sorption. Many kinds of microbes are present, freely floating in water or attached to a substratum, such as macrophytes and sediment, and they grow using different carbon and energy sources.23) Algae and protozoa can absorb and metabolize pesticides,24) but bacteria and fungi contribute more to pesticide dissipation through their ubiquity.3,8,18) Bacteria are more diversely populated, with their activity depending on the habitat,25) while fungi tend to grow on a substratum, such as detritus, rather than in water.26,27) Bacteria metabolize pesticides via various enzymatic reactions,28) and extra-cellular enzymes such as laccases,29) may participate in their fungal degradation. In addition to an active uptake by microbes, pesticides are adsorbed onto surfaces of microbes and biofilm, a gel-type complex mixture of microbial cells, detritus, and extra-cellular polymeric substances (EPS).30,31) Since the constituents of EPS, such as polysaccharides and proteins, have various functional groups, they provide adsorption sites for various pesticides via hydrophobic, electrostatic, and hydrogen-bonding interactions. Microbial degradation can be studied by using the experimental designs prescribed in avalable guidelines.5–7) The contribution of hydrolysis should be examined using a sterile control. Either a microbial community concentrated by a filtration technique or microbes isolated from surface water may be conveniently used to identify potential transformation paths. The relative contribution of each process highly depends on the chemical structure of the pesticide. Through the dissipation of parathion (40) and malathion (49) in estuarine water, sunlight photolysis was found to be much less important than hydrolysis and microbial degradation, respectively.32) N-Methyl carbamates dissipated mainly via hydrolysis and photolysis in lake waters, while bacterial and fungal degradation played more significant roles for N-phenyl carbamates.21) By comparison with sterile controls, a high contribution of biotic processes (>70%) was reported for chlorothalonil (4) and pendimethalin (55) in
12 T. Katagi
the nursery recycling pond water but less (20–50%) for organophosphorus pesticides (OPs) possibly due to the less relevant microbial activity in high salinity.33) Outdoor monitoring of pesticide dissipation is another approach to estimate the relative contribution of each process. Through a series of outdoor studies using open and closed bottles containing intact or filtered (0.45 µm) river water, the contribution of each process was assessed for thirteen OPs.34) Sorption to suspended particles retarded the dissipation of hydrophobic OPs. The degradation under sterile conditions was not available, but the reported hydrolysis data17) showed the importance of biodegradation and photolysis with much less contribution of volatilization. A recent study on four OPs, conducted at controlled temperatures with sterilization, clarified that dominant process of dissipation is highly dependent on their chemical structures.35) More detailed monitoring of pentachlorophenol (2) dissipation was conducted outdoors in experimental flowing channels including aquatic plants and unglazed ceramic tiles for biofilm development.36) Insignificant volatilization of (2) from water and attenuation of photolysis with the water depth were confirmed. The dominant process was biodegradation (26–46%) by microbes attached to macrophytes, biofilm, and sediment. For persistent and hydrophobic pesticides, dilution or adsorption to the bottom sediment was found to be more significant in farm ditches.16)
Study Design of Aerobic Microbial Transformation Excellent reviews are available on various biodegradation methods, each of which was developed to simulate a specific environmental situation.37–39) The important experimental factors are the origin of the inoculum, the concentration of a chemical, the inorganic or organic nutrients used, and the system conditions such as aerobicity, temperature, mixing, and the duration of incubation. The changes in DOC, total organic carbon (TOC) and biochemical oxygen demand (BOD), and formation of carbon dioxide are generally used as a substrate-independent index to examine the ultimate biodegradation of a chemical, while substrate-specific methods such as gas (GC) and liquid (LC) chromatography are applied to assess the primary biodegradation. In contrast to screening and inherent biodegradability tests at high concentrations of a chemical and biomass (activated sludge or sewage plant secondary effluent), the microbial process of a chemical in surface water should be examined at an environmentally relevant ppb–ppm level using microbial community therein. From this viewpoint, modified die-away methods have been proposed using a concentrated microbial community by filtration of river water and validated for various simple aromatics with TOC as a degradation index.40,41) By using these methods, the biodegradation of various pesticides in Japanese river and pond waters at 20–30°C was examined using GC in the presence of 0.1–0.2% organic nutrients.42–44) Their biodegradation half-lives significantly varied among surface waters sampled at different sites and seasons. The nutrient effects in this system
Journal of Pesticide Science
were examined through the biodegradation of chlornitrofen (57) using river waters.45) The moderate biodegradation of (57) (80% after 18 days) was not affected by the addition of a mixture of inorganic salts, while the presence of 0.2% polypeptone resulted in complete degradation after 5 days. To simulate the biodegradation of pesticides at an environmentally relevant concentration, Cripe et al.46) developed a modified river die-away test under shaking using fresh river water without additional nutrients and examined the effect of sediment (0.5 g L−1). A comparison of dissipation under non-sterile and sterile conditions showed that the degradation of parathionmethyl (41) was markedly enhanced by sediment-associated microbes while partition to sediment predominated in the dissipation of more hydrophobic methoxychlor (9). Sediment-mediated biodegradation of fenthion (43) was dominant in salt-marsh waters by using the same method, as compared with hydrolysis and biodegradation in water.47) The usage of radio-labeled (14C) compound gives information on a product distribution in a test system as well as the extent of mineralization. Dissociative pentachlorophenol (2) was partly adsorbed to sediment particles (10–20%) in river waters, but the presence of sediment enhanced the degradation of (2) with a shorter acclimation period, showing the involvement of sediment-associated microbes.48) The slower degradation of 2,4-d (10) but with shorter acclimation periods than those of (2) and no mineralization of atrazine (52) in the same river water indicated a different population of microbes specifically degrading each pesticide.49) There are three test guidelines available for pesticides, and the details are summarized in Table 1. In all cases, freshly collected surface water and sediment are used, and the decline of a pesticide is monitored to determine the degradation rate constant, which can be used in modeling for assessment. OECD 3095) requires two low concentrations of a pesticide, most likely to examine the possible concentration effect on degradation, as discussed in more detail later. Agricultural Canada T-1-2556) characteristically requires that the study be carried out under illumination with a fluorescent light unless a pesticide is photo-labile. U.S. EPA OPPTS 835.31707) recommends the collection of water 6 cm below the surface, which may exclude unexpectedly high adsorption or degradation by neustons and surface microlayer constituents.4) The top sediment should be collected to represent the effect of aerobic microbes as well as biofilm. Identification of major metabolites with their formation rates is important to assess the impact on aquatic species.
Kinetic Analysis The apparent biodegradation rate constant is estimated from the decline curve of a relevant parameter such as a pesticide concentration, assuming various kinetic models.38,50,51) When an acclimation or lag period (Ta≥0) is observed before the initiation of apparent biodegradation, kinetic analysis using “t−Ta” as a time scale should be conducted. The simplest expression on the biodegradation of a substrate (S) is the pseudo-first-order reaction kinetics below, under the constant concentration of biomass (B).
Vol. 38, No. 1, 10–26 (2013)
Aerobic microbial transformation of pesticides in surface water 13 Table 1. Study design of aerobic degradation
Guideline
OECD 309
OPPTS 835.3170
Agricultural Canada
Simulation Biodegradation
Shake Flask Die-Away Test
Guideline T-1-255
Chemical
Radiolabel or non-radiolabel
Radiolabel or non-radiolabel
Radiolabel
Concentration
Two (differ by a factor of 5–10) ≤0.1 ppm and preferably 50d, 6–12d
—
94
(12)
MCPA
R, E (8.7)
10 ppb, M, 25
8–40d, —
0.02
20
(13)
dichlorprop
R, E (8.7)
10 ppb, M, 25
7–46d, —
0.02
20
(16)
propanil
R (8–9)
1 ppm, M, -
3–4d, 3–4d
—
6NW (7–8)
0.1 and 1 ppm, S,17–22
ca.1d,
