Journal of Applied Phycology 10: 547–554, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
547
Effect of thiobencarb on growth and photosynthesis of the soil alga Protosiphon botryoides (Chlorophyta) Hamed M. Eladel1,2 , William J. Henley1 & Imam A. Kobbia3 1 Department
of Botany, Oklahoma State University, Stillwater, OK 74078, U.S.A. Department, Faculty of Science, Benha University, Egypt 3 Botany Department, Faculty of Science, Cairo University, Egypt 2 Botany
(∗ Author for correspondence and reprints: fax +1-405-744-7074; e-mail
[email protected]) Received 1 August 1998; revised 16 September 1998; accepted 17 August 1998
Key words: herbicide, green alga, growth, nutrients, photosynthesis, Protosiphon botryoides, respiration, Thiobencarb Abstract The effects of the herbicide thiobencarb (Saturn) were tested on the growth and physiology of the chlorophyte Protosiphon botryoides isolated from an Egyptian paddy. Assays were conducted using 16-day batch cultures. Chlorophyll and dry weight biomass yields were significantly reduced at 2–3 mg L−1 thiobencarb, and dark respiration increased and protein decreased significantly at 3 mg L−1 . Reductions in exponential specific growth rate (µ) were generally small, but in some cases significant. Thiobencarb also slightly, but significantly, reduced the 77 K fluorescence parameter Fv /Fm , an indicator of maximum photosynthetic efficiency. No consistent dosedependent changes occurred in chlorophyll per unit dry weight, total carbohydrate or gross photosynthetic capacity. Whereas half of the added thiobencarb was recovered from control (uninoculated) medium, it was largely absent from cells and culture medium after sixteen days, indicating biodegradation by the alga or associated bacteria. P. botryoides recovered fully within sixteen days following subculture in thiobencarb-free medium. Independently varying phosphate and nitrate nine-fold had no clear effect on the sensitivity of P. botryoides to thiobencarb.
Introduction The widespread use of herbicides in modern agriculture might adversely affect algal flora. Thiobencarb [S-4-chlorobenzyl diethylthiocarbamate], which has Saturn as principle trade name, is a thiocarbamate herbicide with moderate water solubility (30 mg L−1 at 20 ◦ C). It is widely used for weed control in paddy fields due to its low persistence and high effectiveness. It interferes with protein synthesis and inhibits photosynthesis (Tomlin, 1994). Thiobencarb undergoes volatilization, adsorption, chemical and microbiological transformations in the environment (Ishakawa et al., 1997; Ross & Sava, 1986). The recommended field application dose in terms of active ingredients is approximately 4 kg ha−1 , or 40 mg L−1 for a 10-cm deep paddy (Beste et al., 1983).
Many reports indicate herbicide effects on soil algal growth, photosynthesis, nitrogen fixation, biochemical composition, and metabolic activities (e.g. Mishra & Pandayt, 1989; Bhunia et al., 1991), as well as degradation and removal of herbicides by algae (Stratton, 1984). various algae have been used to study thiobencarb herbicide mode of action, and to reveal its toxicological effects. Yoo (1979) investigated the effects of thiobencarb on the growth, survival and succession of green algae in order to better understand the interaction of thiobencarb herbicide and the primary producers of aquatic ecosystems. Thiobencarb was toxic to the growth of the cyanobacteria Anabaena oryza and Nostoc calcicola (Mahmoud et al., 1988), and was more toxic than knockweed on the growth of A. variabilis Ahluwalia (1988). Thiobencarb was lethal to Nostoc sp. at concentrations ranging from 6
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548 to 8 mg L−1 (Mishra & Pandey, 1989). The inhibitory effect of thiobencarb on the growth, nitrogen fixation, chlorophyll a content and heterocyst formation in a mixed culture of Anabaena, Nostoc and Oscillatoria was quite marked at 55 mg L−1 (Zargar & Dar, 1990). Thiobencarb concentrations ranging from 2 to 6 mg L−1 decreased DNA, RNA, and total protein content and growth of N. muscorum (Bhunia et al., 1991). At low concentrations thiobencarb significantly increased the level of carbohydrates in cells of A. osillarioides and N. Kihlmani, while at higher concentrations carbohydrate content was significantly reduced (Mansour et al., 1994). Susceptibility to thiobencarb differs among the planktonic algae. Among green algae, strains of Selenastrum capricornutum were much more sensitive than Chlorella vulgaris (Kasai & Hatakeyama, 1993), and Scenedesmus acutus was more sensitive than C. saccharophila, while the cyanobacterium Pseudanabaena galeata was intermediate between the latter two species (Sabater & Carrasco, 1996). Ten mg L−1 thiobencarb completely inhibited growth of all Anabaena spp., but was nonlethal to Nostoc spp. (Nagpal & Goyal, 1992). Thiobencarb had no pronounced effect on growth and nitrogen fixation on some heterocystous cyanobacteria using half, full or double rate of the standard field dose (Kolte & Goyal, 1992). Most studies have tested the effect of herbicide in a single medium. However, toxicity to algae may be affected by many environmental factors. The toxicity of carbofuran and hexachlorocyclohexane (HCH) pesticides to Nostoc muscorum was reduced when provided with higher than normal phosphate, nitrate or chloride (Kar & Singh, 1979). The addition of carbon sources, including glucose, acetate and some amino acids (glutamine, arginine, serine, tryptophan), enhanced protection against thiobencarb toxicity to N. linckia (Mishra & Pandey, 1989). At LC50 of dimethoate, the growth rate of Anabaena doliolum increased with increasing concentrations of nitrate, phosphate or sulfate, and the LC50 increased at higher nutrient concentrations (Mohapatra & Mohanty, 1992). Both light and nutrients influenced molinate toxicity and its effects on chlorophyll a, biliprotein and protein content (Guoan et al., 1997). The removal of herbicides from the environment is of great importance. Algae have been used to remove herbicides from aquatic environments, and herbicides are known to be metabolized by various algae (Valentine & Bingham, 1973). The aim of this study was to examine the growth, uptake of herbicide
and effects of nitrate and phosphate on the sensitivity of the soil chlorophyte Protosiphon botryoides to thiobencarb.
Materials and methods Organism and culture conditions Protosipon botryoides (Kütz.) Klebs, a simple siphonous chlorophyte, was isolated from paddy field soil samples at Qalubia, Egypt. Although farmers in this area do not routinely apply herbicides, we cannot be absolutely sure that the isolate had been never been exposed to herbicides. Isolation and purification was made by dilution and plating technique. Cultures were grown in 250-mL flasks containing 100 mL Bold’s basal medium (BBM; Bischoff & Bold, 1963), and incubated in a controlled growth camber at 24 ± 2 ◦ C and 70 µmol m−2 s−1 photosynthetically active radiation (PAR), provided by cool white fluorescent lamps set on 16:8-light:dark photoregime for 16 d. All cultures were shaken twice daily to prevent cells from clumping. Sterile technique was used at all times, but axenic status was not routinely monitored, and thus we must assume that bacteria could have been present. Thiobencarb (technical grade, 95%) was obtained from KZ Company (Egypt). Concentrated herbicide stock was prepared in acetone. Aliquots of the stock were added to culture flasks, and the acetone was completely evaporated. Medium was then added and flasks were left for 1 d to obtain aqueous solutions of 0, 1, 2 or 3 mg L−1 . All cultures (three per treatment) received identical inocula and were incubated under the prescribed growth conditions. The first experiment considered the effect of thiobencarb concentration on Protosiphon in standard BBM, and followed algal uptake of the herbicide, and algal recovery following herbicide removal. In the next two experiments, BBM medium was modified to contain different levels of nitrate (1, 3 or 9 mM) and phosphate (0.6, 1.7 or 5 mM), to study the effect of nutrient availability on the toxicity of thiobencarb to Protosiphon. These experiments were conducted concurrently and standard BBM (3 mM nitrate, 1.7 mM phosphate) served as the control for both experiments. Growth measurements Chlorophyll a (Chl a) was extracted by diluting 0.5 mL sample to 5 mL with N, N-dimethyl formamide (DMF) and storing for 2 h at room temperature in darkness.
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549 Cells were removed by centrifugation, and Chl fluorescence was measured with a Turner 111 fluorometer calibrated with spectrophotometrically quantified Chl a (Shimadzu UV- 160U, 2 nm bandpass). The specific growth rate (µ, d−1 ) was determined for individual cultures by linear regression through Chl data from days 2, 4, 6 and 8 in the exponential growth phase. At the end of the experiments biomass yield was estimated as Chl a and dry weight, and physiological state as photosynthesis, respiration and variable fluorescence. Total protein and carbohydrates were also measured after centrifugation of cells from 10 ml culture at the end of the initial experiment and its recovery period. Proteins were determined by the Bradford method following extraction of cell pellets with 0.1 N NaOH for 30 min (Jones et al., 1989), using bovine serum albumin (BSA) as standard. Total carbohydrate was extracted according Myklestad and Haug (1972), and quantified by the phenol sulfuric acid method using glucose as standard (Dubois et al., 1956). At the end of each experiment, cells were harvested on a preweighed Whatman GF/F filter, washed with distilled water three times, then dried at 105 for 8 h to obtain dry weight. Photosynthetic measurements Photosynthetic light-response (P–I) curves were measured as O2 exchange at 24 ± 2 ◦ C in a Hansatech DW3 water-jacketed, 10 ml polarographic electrode chamber, connected to a National Instruments 12-bit A/D board controlled by custom-written software. The samples were exposed to a series of ten incremented PFDs from darkness to > 1000 µmol photons m−2 s−1 , with at least five PFDs in the linear initial slope region (Henley, 1993). Light was provided from a slide projector, and PFD was incremented automatically at 150 sec intervals with neutral density filters and measured with a LI-COR LI-189 m and LI-1000 quantum sensor located on the rear window of the chamber. Nonnormalized rates of O2 exchange (µmol O2 h−1 ) were automatically calculated in real time by linear regression after each PFD. Rates were normalized to Chl a. Light-saturated gross photosynthesis (Pm ), initial slope (α) and dark respiration (Rd ), and photoinhibition parameter (β) were determined by fitting individual curves to a saturation function (Platt et al., 1980). Fitted parameters for three replicate curves were then averaged. Only a few of the curves exhibited a downturn at high PFD, but the photoinhibition equation was used for all curves for consistency. Es-
Figure 1. Chlorophylll a growth of Protosiphon botryiodes in (◦) control, () 1 mg L−1 , (4) 2 mg L−1 and (♦) 3 mg L−1 thiobencarb in (A) initial treatment and (B) recovery.
timates of α were highly variable and unrelated to experimental treatment, so are not reported. Fluorescence measurements at 77 K A customized, computer-controlled fluorometer was used for measurement of fluorescence induction at 77 K (Henley et al., 1991). Cells were collected on glass-fiber filters, from which a 1-cm disk was cut and appressed directly to the bottom of a quartz rod (1 cm diameter, 10 cm long), and secured with a thin metal cap. After 5 min dark-adaptation, the rod was immersed into liquid N2 for 2 min, and then measurement was started. Thiobencarb residue measurement For thiobencarb residue measurements, 100 mL was withdrawn from each culture on day sixteen. Algal cells were collected on GF/F filters, and thiobencarb
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2.85 ± 0.39 2.55 ± 0.46 2.73 ± 0.27 2.55 ± 0.04 NS
0.16 ± 0.03 0.17 ± 0.04 0.18 ± 0.01 0.18 ± 0.00 NS
∗∗∗
1.81 ± 0.15 1.31 ± 0.08∗∗∗ 1.93 ± 0.15∗∗∗ 0.38 ± 0.04∗∗∗
0.14 ± 0.02 0.22 ± 0.02∗∗ 0.13 ± 0.03 0.06 ± 0.02∗∗
Control 1.0 2.0 3.0 One-way ANOVA RECOVERY Control 1.0 2.0 3.0 One-way ANOVA ∗∗∗
Chl yield mg L−1
µChl d−1
Thiobencarb mg L−1
438 ± 19 427 ± 64 427 ± 64 430 ± 100 NS
∗∗∗
181 ± 15 144 ± 16∗ 95 ± 14∗∗∗ 61 ± 5∗∗∗
DW yield mg L−1
6.5 ± 1.1 6.0 ± 0.4 6.6 ± 1.5 6.1 ± 1.5 NS
∗∗∗
10.0 ± 0.7 9.2 ± 4.1 9.7 ± 0.3 6.3 ± 0.1∗∗∗
Chl mg g−1 DW
40 ± 5 50 ± 9 63 ± 13∗ 44 ± 11 NS
∗
88 ± 4 75 ± 23 77 ± 3 55 ± 5∗
Protein mg g−1 DW
97 ± 29 111 ± 28 83 ± 8 58 ± 20 NS
175 ± 18 178 ± 158 140 ± 59 196 ± 97 NS
Carbohydrate mg g−1 DW
0.80 ± 0.01 0.80 ± 0.01 0.81 ± 0.01 0.81 ± 0.01 NS
∗∗
0.72 ± 0.02 0.66 ± 0.01∗∗ 0.66 ± 0.02∗∗ 0.68 ± 0.01∗
Fv /Fm
195 ± 51 196 ± 10 184 ± 4 225 ± 23 NS
304 ± 36 254 ± 37 219 ± 26 320 ± 95 NS
Gross Pm Chl mol O2 g−1
− 44 ± 46 − 17 ± 9 − 20 ± 12 − 21 ± 28 NS
∗∗
− 43 ± 16 − 50 ± 11 − 54 ± 20 − 108 ± 8∗∗∗
Rd Chl Chl h−1
Table 1. Specific growth rate (µ) over days 2–8, and Chl and dry weight (DW) yield, Chl DW−1 , protein, carbohydrates, Fv /Fm , light-saturated gross photosynthetic capacity (Pm Chl ), and dark respiration (Rd Chl ) on day 16 of thiobencarb treatment and recovery periods. Means ± SD (n = 3). Results of one-way ANOVA and Dunnett’s one-sided comparison of treatments to controls indicate ∗ P < 0.05; ∗∗ < 0.01; ∗∗ P
