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Effects of bensulfuron-methyl and cinosulfuron on growth of four freshwater species of phytoplankton
Chemosphere 46 (2002) 953–960 www.elsevier.com/locate/chemosphere
Effects of bensulfuron-methyl and cinosulfuron on growth of four freshwater species of phytoplankton C. Sabater *, A. Cuesta, R. Carrasco Pesticide Laboratory, Department of Biotechnology, E.T.S.I.A., Polytechnic University of Valencia, Camino de Vera 14, 46022 Valencia, Spain Received 26 April 2001; accepted 17 August 2001
Abstract The acute toxicity of sulfonylurea herbicides bensulfuron-methyl and cinosulfuron was tested on the five species of freshwater phytoplankton: Scenedesmus acutus, Scenedesmus subspicatus, Chlorella vulgaris and Chlorella saccharophila. Herbicide concentrations eliciting a 50% growth reduction over 96 h ðEC50 Þ ranged from 8 to 104 mg/l for cinosulfuron and from 0.015 to 6.2 mg/l for bensulfuron-methyl. The pesticides bensulfuron-methyl, atrazine and benthiocarb were more toxic than cinosulfuron, chlorsulfuron, molinate, fenitrothion and pyridaphenthion in a toxicity study with the same algal species. The transformation of effective concentrations of bensulfuron-methyl and cinosulfuron and other pesticides, obtained from toxicity measurements, into percent of the saturation level in water is used as a first evaluation of potential hazard to aquatic systems. The herbicides cinosulfuron, methyl-bensulfuron, atrazine and chlorsulfuron were more dangerous than the herbicides benthiocarb and molinate and than the insecticides fenitrothion and pyridaphenthion, in a study of hazard evaluation. The two species of Chlorella were more tolerant to both herbicides than the two species of Scenedesmus. A potential environmental hazard of sulfonylurea herbicides to aquatic systems has to be expected even at low environmental concentrations. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Toxicity; Herbicide; Bensulfuron-methyl; Cinosulfuron; Algae
1. Introduction A wide range of pesticides are used to protect agricultural crops. Although the instructions for use of these chemicals aim at minimizing the risk of contamination of aquatic environments, residuals of pesticides can be detected in water courses draining agricultural areas (K€ allqvist and Romstad, 1994). In this way, the sulfonylurea herbicides such as bensulfuron-methyl and cinosulfuron are characterized by broad-spectrum weed control at very low use rates (2–75 g/ha), good crop
*
Corresponding author. Tel.: +34-9-638-77428; fax: +34-9638-77429. E-mail address: [email protected] (C. Sabater).
selectivity, very low acute and chronic animal toxicity and no propensity to bioaccumulate in non-target organisms. All these characteristics make the sulfonylurea herbicides ideal candidates for replacing some of the older herbicides in an effort to reduce the quantity of chemicals used (Pimentel et al., 1991) and to eliminate herbicides with relatively high animal toxicity (Fletcher et al., 1993). This class of herbicides acts through inhibition of acetolactate synthase (specific to plants and microorganisms) and thereby blocking the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. This inhibition leads to the rapid cessation of plant cell division and growth (Brown, 1990). Bensulfuron-methyl [methyl 2-[[[[[(4,6-dimethoxy-2pyrimidinyl) amino] carbonyl] amino] sulfonyl] methyl] benzoate] controls many problem broadleaf weeds and
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sedges in continuously flooded rice, at 30–100 g/ha. In combination with thiocarbamate grass herbicides, bensulfuron-methyl provides total season-long weed control with good crop safety (Beyer et al., 1988; Tomlin, 1997). Cinosulfuron [N-[[(4,6-dimethoxy-1,3,5-triazin-2-yl) amino] carbonyl]-2-(2-methoxyethoxy) benzenesulfonamide] is applied post-emergence to control many weeds in transplanted, direct seeded, wet-sown and watersown, and dry-sown flooded rice crops, at 20–80 g/ha (Tomlin, 1997). Because these herbicides are typically applied to rice fields, contamination of nearby aquatic systems may occur through a variety of mechanisms, including overspray, drift, surface runoff and leaching (Thompson et al., 1992). It is important to assess the adverse impacts these chemicals may have on non-target organisms in aquatic ecosystems. Wetland contamination could result in a die-off of most algal species present, causing complete disappearance of this food source. Alternatively, certain species or groups of algae could be selectively inhibited. Algae and cyanobacteria are known to be comparatively sensitive to many chemicals, and the inclusion of these organisms in test batteries has been shown to improve the capacity of the battery to predict the most sensitive ecosystem responses (Sloof et al., 1983). Furthermore, the importance of these organisms as dominating primary producers in most aquatic ecosystems speaks for their use in test batteries for environmental hazard assessment. An examination of published phytoplankton toxicity data demonstrates that wide differences in sensitivity can occur across phylogenetic groups of phytoplankton (Blanck et al., 1984; K€allqvist and Romstad, 1994; Fairchild et al., 1997). Much research concerning the toxicity of sulfonylurea herbicides on weeds and crops has been reported (Beyer et al., 1988; Fletcher et al., 1993; Dastgheib et al., 1995); there are few reports about their effects on aquatic organisms. The objective of this work was to investigate the effective concentrations of bensulfuron-methyl and cinosulfuron that caused 50% inhibition on growth, in pure cultures, of two wild species of phytoplankton, representatives of Mediterranean wetlands, isolated from Lake Albufera (Valencia, Spain) and two laboratory strains.
2. Materials and methods The chlorophyceae Scenedesmus acutus (Meyens), Scenedesmus subspicatus CCAP 276/22, Chlorella vulgaris Beijerinck and Chlorella saccharophila (Kr€ uger) Migula were selected for the toxicity tests. S. acutus and C. saccharophila were isolated from samples collected at the Albufera lake in Valencia (Spain). S. subspicatus and C. vulgaris were kindly supplied by the Institute of
Freshwater Ecology (Ambleside, UK) and by the Area of Environmental Toxicology (CISA-INIA, Spain), respectively. These four chlorophyceae were grown in a medium recommended by the ASTM (1993). The stock cultures were maintained in a liquid medium at a temperature of 22 2 °C and a light intensity of 1100 lux on a 12-h light-dark cycle. Bensulfuron-methyl (99.4%, analytical standard) and cinosulfuron (98%, analytical standard) were obtained from DuPont Iberica and Ciba-Geigy (Valencia, Spain). Bensulfuron-methyl (pure) is a solid with molar mass 396.4 and with melting point 185–188 °C and vapor pressure 17 nPa (25 °C); its Kow is 155 (pH 5) and 4.1 (pH 7) and its pKa 5.2; its solubility (25 °C) in water is 2.9 mg/l at pH 5 and 120 mg/l at pH 7; its solubility (20 °C) in acetonae, acetonitrile, dichloromethane and hexane is 1.38, 5.38, 11.7 and 0.31 mg/l, respectively. Cinosulfuron (pure) is a colorless crystalline powder with a molar mass of 413.4; water solubility of cinosulfuron (20 °C) is 18 mg/l (pH 2.5), 82 mg/l (pH 5) and 3.7 g/l (pH 7); its solubility (20 °C) in dichloromethane and dimethyl sulfoxide is 95 and 320 g/kg, respectively, and is slightly soluble in common organic solvents; its melting point and vapor pressure (20 °C) is 144.6 °C and 100 pPa, respectively; its Kow at (25 °C) is 2.04 (pH 2.1) and 0.63 (pH 7) and its pKa 4.72 (Worthing and Hance, 1991; Tomlin, 1997). The inhibition test was conducted in accordance with the ASTM (1993) protocol. The organisms were exposed to various concentrations of bensulfuron-methyl and cinosulfuron for 96 h. Growth of cultures was measured by the turbidity at 750 nm wavelength using a spectro€ rdog and Kuivasniphotometer (Beckman DUâ -70) (O emi, 1989) at 24, 48, 72 and 96 h after the start of the test. After 96 h, growth rates were quantitatively determined using the exponential model y ¼ eaþlx [where y ¼ the population estimate, x ¼ time, a ¼ integration constant and l ¼ slope or growth rate]. ANOVA and Student Newman–Keuls multiple range test were employed to determine if treatments were significantly different from each other (Reish and Oshida, 1987). Results were deemed significantly different at the level P 6 0:05. EC10 (0–96 h) and EC50 (0–96 h) values with 95% confidence limits were estimated by the linear regression of probit of percentage growth on the log dose of bensulfuron-methyl and cinosulfuron (Newman, 1995). In this study, EC10 and EC50 are the concentrations of herbicides, derived by the method of calculation ‘‘comparison of areas under growth curves’’, which results in a 10% and a 50% growth reduction relative to the control values, at 96 h, respectively (ASTM, 1993). All statistical analyses were performed using the commercial software packages (STATGRAPHICS, 1994) (STSC, Rockville, Md.) and SAS (1988) (SAS Institute, Cary). Further details of test protocols are provided in Sabater and Carrasco (1996).
C. Sabater et al. / Chemosphere 46 (2002) 953–960
At the beginning and at the end of the assays, herbicide concentrations in test solutions were determined by HPLC; the results were analyzed statistically and used for calculating the toxicities of bensulfuron-methyl and cinosulfuron. For the extraction and determination of herbicides the test culture samples (25 ml) were filtered, acidified with HCl 1 N and extracted with three 20 ml portions of dichloromethane by shaking in a separatory funnel. The combined hexane extracts were filtered through NaSO4 to remove residual water and evaporated to dryness and the residue was dissolved in 5–10 ml of acetonitrile/NH3 0.024 M (30/70, v/v) and analyzed by HPLC with absorbance detection at 220 nm. The column was a 15 cm Shodex DS-613 RS-pak and the mobile isocratic phase was acetonitrile/aqueous phosphate (103 M) acidified with phosphoric acid (pH 4.6), (60:40, v/v) (Galletti et al., 1995; Carrasco, 1999). Approximated retention time for bensulfuron-methyl is 5:71 0:17 min and for cinosulfuron is 3:72 0:04 min. The recovery of bensulfuron-methyl and cinosulfuron was in the range between 93 2% and 90 5%, respectively.
3. Results and discussion Experimental bensulfuron-methyl and cinosulfuron exposure concentrations measured by GC (no nominal values) are shown (Tables 1 and 2). The concentrations of bensulfuron-methyl that caused significant effects ðP < 0:05Þ on the algal growth with respect to control values ranged from 5.0 to 18.9 mg/l for C. saccharophila, from 0.21 to 15.5 mg/l for C. vulgaris, from 0.0083 to 0.32 mg/l for S. acutus and from 0.035 to 3.6 mg/l for S. subspicatus. The concentrations of cinosulfuron that caused significant effects ðP < 0:05Þ on the algal growth were markedly higher, between 41.5 and 358.0 mg/l for C. saccharophila, between 62.3 and 355.5 mg/l for C.
955
vulgaris, between 3.6 and 98.8 mg/l for S. acutus and between 11.6 and 150.7 mg/l for S. subspicatus. Growth of the four species of phytoplankton under different exposures of bensulfuron-methyl and cinosulfuron to that of controls was compared (Tables 1 and 2). A broad range of sensitivity within species was observed for both herbicides. Reduction in growth rates was observed with an increase of bensulfuron-methyl and cinosulfuron concentrations. At 5.0, 0.21, 0.0083 and 0.035 mg/l of bensulfuron-methyl the growth of C. saccharophila, C. vulgaris, S. acutus and S. subspicatus was significantly inhibited. However, doses of cinosulfuron greater than 41.5 mg/l for C. saccharophila, 62.3 mg/l for C. vulgaris, 3.6 mg/l for S. acutus and 11.6 mg/l for S. subspicatus were necessary for producing similar bensulfuron-methyl effects on the growth of these species. In contrast to Chlorella species, whose growth was strongly inhibited by concentrations of bensulfuron-methyl of 15.5–18.9 mg/l (lethal concentrations), Scenedesmus species were highly sensitive at lower concentrations of this herbicide, 0.32–3.6 mg/l. No inhibition of the growth of Chlorella species was observed at 26.1 and 36.8 mg/l of cinosulfuron, respectively. Conversely, strong inhibition of S. acutus growth was observed at 35.9 mg/l of this herbicide. Based on their 96-h EC10 and EC50 values, the four species of algae responded very differently to bensulfuron-methyl and cinosulfuron. Scenedesmus species were more sensitive to both herbicides than were Chlorella species. S. acutus and S. subspicatus both proved to be very sensitive to bensulfuron-methyl, with 10% and 50% inhibition of growth at bensulfuron-methyl concentrations of 0.0052–0.0095 and 0.015–0.034 mg/l, respectively. The 96 h EC10 and EC50 values of cinosulfuron for algae assayed were markedly higher than were for bensulfuron-methyl, ranging from 1.8 to 36 mg/l for Scenedesmus species and from 30 to 104 mg/l for Chlorella species. The toxicities of bensulfuron-methyl and
Table 1 Average specific growth rates (la ) of the four species of phytoplankton treated with bensulfuron-methyl C. saccharophila
C. vulgaris
S. acutus
S. subspicatus
mg/l
l
R2
mg/l
l
R2
mg/l
l
R2
mg/l
l
R2
0.0 2.3b 5.0 5.9 7.0 18.9c
0.77 0.077 0.075 0.073 0.066 –
0.93 0.93 0.96 0.98 0.99 –
0.0 0.010b 0.21 1.6 5.7 15.5c
0.063 0.063 0.059 0.056 0.047 –
0.98 0.97 0.97 0.98 0.98 –
0.0 0.0017b 0.0083 0.017 0.034 0.32c
0.059 0.059 0.057 0.053 0.039 –
0.97 0.97 0.97 0.98 0.92 –
0.0 0.0022b 0.035 0.053 0.15 3.6c
0.059 0.059 0.056 0.049 0.044 –
0.92 0.92 0.99 0.99 0.99 –
R2 : Correlation coefficient. a Growth rates (l) were determined using the exponential model, y ¼ eaþlx [y ¼ the population estimate, x ¼ time, a ¼ integration constant and l ¼ slope or growth rate]. b Highest concentrations tested without significant effects ðP < 0:05Þ on the algal growth relative to control values. c Lethal concentrations.
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C. Sabater et al. / Chemosphere 46 (2002) 953–960
Table 2 Average specific growth rates (la ) of the four species of phytoplankton treated with cinosulfuron C. saccharophila
C. vulgaris 2
S. acutus 2
S. subspicatus 2
mg/l
l
R
mg/l
l
R
mg/l
l
R
mg/l
l
R2
0.0 26.1b 41.5 65.4 112.9 358.0c
0.077 0.077 0.075 0.072 0.067 –
0.96 0.94 0.95 0.97 0.98 –
0.0 36.8b 62.3 93.9 112.8 355.0c
0.057 0.057 0.056 0.051 0.039 –
0.99 0.99 0.99 0.99 0.98 –
0.0 0.52b 3.6 15.0 35.9 98.8c
0.059 0.059 0.056 0.051 0.036 –
0.95 0.96 0.98 0.99 0.97 –
0.0 2.5b 11.6 36.4 78.0 150.7c
0.060 0.060 0.059 0.055 0.047 –
0.92 0.91 0.97 0.99 0.99 –
R2 : Correlation coefficient. a Growth rates (l) were determined using the exponential model, y ¼ eaþlx [y ¼ the population estimate, x ¼ time, a ¼ integration constant and l ¼ slope or growth rate]. b Highest concentrations tested without significant effects (P < 0:05) on the algal growth relative to control values. c Lethal concentrations.
Table 3 Algal EC10 and EC50 (0–96 h) values of bensulfuron-methyl and cinosulfuron with 95% confidence limits for the four species Test species
Herbicides Cinosulfuron (mg/l)
C. saccharophila C. vulgaris S. acutus S. subspicatus
Bensulfuron-methyl (mg/l)
EC10
EC50
EC10
EC50
30 (28–34) 38 (31–46) 1.8 (0.9–2.3) 10.2 (9.5–13.0)
104 (89–128) 96 (94–98) 8 (4–13) 36 (32–40)
2.7 (1.4–3.4) 0.40 (0.36–0.44) 0.0052 (0.0041–0.0068) 0.0095 (0.0088– 0.0130)
6.2 (6.0–6.3) 2.2 (1.8–2.6) 0.015 (0.014–0.017) 0.034 (0.031–0.037)
cinosulfuron to our species of phytoplankton and toxicity data published for several species of phytoplankton with another sulfonylurea herbicides have shown that the variation in sensitivity may be considerable. Blanck et al. (1988) showed that the range in susceptibility among species to chemicals extended over more than three orders of magnitude. Ninty-six-hour EC50 values of bensulfuron-methyl obtained in our study for C. saccharophila and C. vulgaris have been 2.2 and 6.2 mg/l, respectively. These values are higher than the 96-h EC50 of 0.17 mg/l found by Liping Wei et al. (1998) for growth inhibition in Chlorella pyrenoidosa exposed to bensulfuron-methyl. This value of 0.17 mg/l is, at the same time, greater than bensulfuron-methyl 96-h EC50 of 0.015 mg/l for S. acutus and than bensulfuron-methyl 96-h EC50 of 0.034 mg/l for S. subspicatus (Table 3). Metsulfuron-methyl, chlorsulfuron, triasulfuron and tribenuron-methyl affected growth and reproduction of Chlorella fusca at concentrations between 0.04 and 4 mg/l (Fahl et al., 1995) and Hartnett et al. (1987) observed growth inhibition of Chlamydomonas reinhardtii on agar plates by sulfometuron-methyl at 0.36 mg/l or greater. EC50 values for the effect of chlorsulfuron on the growth rate of several phytoplankton species ranged from 0.15 to 60 mg/l, a total range factor of 400 (Dyer et al.,
1982; K€ allqvist and Romstad, 1994; Carrasco and Sabater, 1997). The macrophyte Lemna minor was highly sensitive to chlorsulfuron (0.7 lg/l; 96-h EC50 ) and metsulfuron ð0:4 lg=l; 96-h EC50 Þ, whereas the green algae Selenastrum capricornutum was 100-fold sensitive (chlorsulfuron, 135 lg=l 96-h EC50 ; metsulfuron, 190 lg=l 96-h EC50 ) (Fairchild et al., 1997). Although a high degree of variability was observed by these authors among the algal species studied, the majority of sulfonylureas EC50 values were in the range from 0.08 to 54 mg/l. However, in our study we found an EC50 for bensulfuron-methyl in Scenedesmus species of 0.015–0.034 mg/l and an EC50 for cinosulfuron in Chlorella species of 96–104 mg/l, which were out of the mentioned range found in the bibliography. The significance of the large interspecies variation in sensitivity among phytoplankton species is not obvious. Exposure of a natural phytoplankton community to a toxic chemical will tend to extinguish the most sensitive species. Because of the generally high reproduction potential of phytoplankton, other species will rapidly replace those that disappear and the standing crop and level of primary production may remain merely unchanged (K€ allqvist and Romstad, 1994). The new community will have a higher tolerance to the specific pollutant, as has been shown experimentally by Blanck
C. Sabater et al. / Chemosphere 46 (2002) 953–960
et al. (1988), who introduced the term ‘‘pollution induced community tolerance’’ (PICT) for this phenomenon. Environmental concentrations of bensulfuron-methyl and cinosulfuron of 0.007–0.025 and 0.003–0.03 mg/l, respectively, have been reported in flooded paddy fields (Allievi, 1996). Concentrations of bensulfuron-methyl are much lower than toxicity values of 0.40 and 6.2 mg/l obtained for Chlorella species and are near to the EC50 of 0.034 mg/l for S. subspicatus but exceeded EC10 values for Scenedesmus species and EC50 value for S. acutus (Table 3), as determined in this study. With regard to ecotoxicological risk assessment for the development of Scenedesmus species under environmental conditions the NOEC and EC10 values (0.0017–0.0095 mg/l) were in the same range as the concentration levels of bensulfuronmethyl found in flooded paddy fields (Allievi, 1996) and as the concentration levels of 0.0003–0.0084 mg/l found by Sabater et al. (2000) at different points in waters from Lake Albufera (Valencia, Spain) in spring and summer of 2000; so the use of bensulfuron-methyl may cause harmful effects on the long-term development of S. acutus and S. subspicatus natural populations. The loss of a few, particularly sensitive, phytoplankton species from a community containing hundreds of species may not be considered significant, as long as the function of the community remains unchanged. However, this may be one of the most sensitive reactions of an ecosystem to a pollution stress, and thus be an important indication of the potential of ecologically harmful effects (K€allqvist and Romstad, 1994). A potential environmental hazard of sulfonylurea herbicides to aquatic systems has to be expected even at
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low environmental concentrations but the ecological fate of bensulfuron-methyl may be changed substantially under several conditions. In rice paddy water, microbial degradation is an important factor in the breakdown of bensulfuron-methyl (Langeland and Laroche, 1994). Chemical hydrolysis was often considered as secondary, but its occurrence must not be completely neglected. In an evaluation of the risk for the receiving surface waters for River Po system (Vighi et al., 1996) the disappearance of bensulfuron-methyl was very rapid, among the water applied pesticides in paddy fields, and their risk was considered absent or negligible throught year with the exception of the month of May. Liping Wei et al. (1998) found that bensulfuron-methyl can be degraded and so would decrease fastly their concentrations into the aquatic media which lead to a rapid decrease in toxicity to the green algae. Toxicity data were further examined for fenitrothion and other pesticides with same algal species, by normalizing EC50 values to water solubility and molar mass by using the percent saturation approach (Gaggi et al., 1995). In this way a first-step evaluation of the hazard to aquatic systems is produced, indicating how far the effective concentration is from the maximum possible in water (Table 4). The combined use of the effective concentrations with percent saturation leads to two results: a classical toxicity ranking and a preliminary hazard classification for aquatic contaminants. Three response groups were observed in Chlorella species, the first about 0.0063–0.25% saturation for clorsulfuron, cinosulfuron, bensulfuron-methyl and atrazine, the second about 1.6–4.3% saturation for molinate and
Table 4 Hazard evaluationa of several compounds to freshwater algae Pesticide Atrazine Benthiocarb Cinosulfuron Chlorsulfuron Fenitrothion Bensulfuron-methyl Molinate Pyridaphenthion a
Test species C. saccharophila
C. vulgaris
S. acutus
S. subspicatus
0.085b 13.4e 0.25 0.0063g 27.1h 0.13 1.6i 18.9j
0.031c 8.4f 0.23 – 56.6h 0.046 4.3i 8.9j
0.0016b 0.057e 0.019 0.00003g 4.0h 0.00032 0.099i 1.5j
0.021d 0.54f 0.086 – 6.2h 0.00072 0.072i 1.3j
EC50 values were transformed into percent saturation, referred to the solubility in water of the chemical as subcooled liquid (Gaggi et al., 1995). b Data from Carrasco and Sabater (1997). c Data from Larsen et al. (1986). d Data from Kirby and Sheahan (1994). e Data from Sabater and Carrasco (1996). f Data from Sabater et al. (2000). g Data from Sabater and Carrasco (1997). h Data from Sabater and Carrasco (2001b). i Data from Sabater and Carrasco (1998). j Data from Sabater and Carrasco (2001a).
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the third about 8.4–56.6% saturation for fenitrothion, pyridaphenthion and benthiocarb. In Scenedesmus species were also observed three response groups, the first about 0.00003–0.0016% saturation for clorsulfuron, bensulfuron-methyl and atrazine, the second about 0.019–0.54% saturation for molinate, benthiocarb and cinosulfuron and the third about 1.3–6.2% saturation for fenitrothion and pyridaphenthion. A comparison of the relative sensitivity of Chlorella and Scenedesmus species to the seven pesticides is presented in Figs. 1–4. Data points that fall on the line (i.e., slope ¼ 1) indicate equivalent sensitivity of the two species to a given chemical. Data that fall to the right of the line indicate that C. vulgaris or S. subspicatus are more sensitive; alternatively, data that fall to the left of the line indicate that C. saccharophila or S. acutus are more sensitive. The collective data indicate that C. vulgaris and S. acutus were more sensitive to pesticides (Figs. 1–4). A large degree of variation in sensitivity among species to both sulfonylureas was obvious in our study, with the growth of same species being completely unaffected by concentrations that were phytotoxic to other species. The two species of Chlorella were considerably more tolerant than two species of Scenedesmus. S. acutus was the most sensitive alga and C. saccharophila the least
Fig. 3. Hazard evaluation of seven pesticides on S. acutus and S. subspicatus.
Fig. 4. Toxicity of seven pesticides on S. acutus and S. subspicatus.
sensitive. The pesticides methyl-bensulfuron, atrazine and benthiocarb were more toxic than cinosulfuron, chlorsulfuron, molinate, fenitrothion and pyridaphenthion in a toxicity study with the same algal species but the herbicides cinosulfuron, bensulfuron-methyl, atrazine and chlorsulfuron were more dangerous than the herbicides benthiocarb and molinate and than the insecticides fenitrothion and pyridaphenthion, in a study of the hazard evaluation. Fig. 1. Hazard evaluation of seven pesticides on C. vulgaris and C. saccharophila.
Acknowledgements This work was supported by a grant from ‘‘Comisi on Interministerial de Ciencia y Tecnologia’’ (1FD97-0963C03-03) and from ‘‘Generalitat Valenciana’’ (GV-DAG-01-154-96).
References
Fig. 2. Toxicity of seven pesticides on C. vulgaris and C. saccharophila.
Allievi, L., 1996. The influenze of herbicides on soil microorganisms and the case for sulfonylureas. Biol. Ital. 26, 20–24. ASTM, 1993. Standard guide for conducting static 96-h toxicity tests with microalgae, E 1218-90. Beyer, E.M., Duffy, M.J., Hay, J.V., Schlueter, D.D., 1988. Sulfonylureas. In: Kearney, P.C., Kaufman, D.D. (Eds.),
C. Sabater et al. / Chemosphere 46 (2002) 953–960 Herbicides, Chemistry, Degradation and Mode of Action, vol. 3. Marcel Dekker, New York, pp. 117–1189. Blanck, H., Wallin, G., W€angberg, S.A., 1984. Species-dependent variation in algal sensitivity to chemical compounds. Ecotoxicol. Environ. Saf. 8, 339–351. Blanck, H., W€ angberg, S.A., Molander, S., 1988. Pollution induced community tolerance – a new ecotoxicological tool. In: Pratt Jr., J., Cairns, J.R. (Eds.), Functional Testing of Aquatic Biota for Estimating Hazard of Chemicals. ASTM STP 988, Amer. Soc. Test. Mater., Philadelphia, pp. 219– 230. Brown, H.M., 1990. Mode of action, crop selectivity, and soil relation of the sulfonylurea herbicides. Pestic. Sci. 29, 263–281. Carrasco, J.M., Sabater, C., 1997. Toxicity of atrazine and chlorsulfuron to algae. Toxicol. Environ. Chem. 59, 89–99. Carrasco, R., 1999. Biodegradation and photodecomposition of pesticides in aquatic systems. Thesis Doctoral, Politechnic University of Valencia, Biotechnology Department, Spain. Dastgheib, F., Andrews, M., Morton, J.D., Barnes, M.F., 1995. Mode of action of chlorsulfuron in a sensitive wheat (Triticum awstivum) cultivar: primary and secondary effect on nitrogen assimilation. Ann. Appl. Biol. 127, 125– 135. Dyer, W.E., Fay, P.K., Thompson, R.H., 1982. Developing a bioassay for chlorsulfuron residues in Montana soils. Proc. Western Soc. Weed Sci. 35, 74. Fahl, G.M., Kreft, L., Altenburger, R., Faust, M., Boedeker, W., Grimme, L.H., 1995. pH-dependent sorption, bioconcentration and algal toxicity of sulfonylurea herbicides. Aquat. Toxicol. 31, 175–187. Fairchild, J.F., Ruessler, D.S., Haverland, P.S., Carlson, A.R., 1997. Comparative sensitivity of Selenastrum capricornutum and Lemna minor to sixteen herbicides. Arch. Environ. Contam. Toxicol. 32, 353–357. Fletcher, J.S., Pfleeger, T.G., Ratsch, H., 1993. Potential environmental risks associated with the new sulfonylurea herbicides. Environ. Sci. Technol. 27, 2250. Gaggi, C., Sbrilli, G., Hasab El Naby, A.M., Bucci, M., Duccini, M., Bacci, E., 1995. Toxicity and hazard ranking of s-triazine herbicides using microtox, two green algal species and a marine crustacean. Environ. Toxicol. Chem. 14, 1065–1069. Galletti, G.C., Bonetti, A., Dinelli, G., 1995. High-performance liquid chromatographic determination of sulfonylureas in soil and water. J. Chromatogr. A 692, 27–37. Hartnett, A.C., Newcomb, J.R., Hodson, R.C., 1987. Mutations in Chlamydomonas reinhardtii confering resistance to the herbicide sulfometuron methyl. Plant Physiol. 85, 898– 901. K€ allqvist, T., Romstad, R., 1994. Effects of agricultural pesticides on planktonic algae and cyanobacteria, examples of interspecies sensitivity variations. Norw. J. Agric. Sci. 13, 117–131. Kirby, M.F., Sheahan, D.A., 1994. Effects of atrazine, isoproturon, and mecoprop on the macrophyte Lemna minor and the alga Scenedesmus subspicatus. Bull. Environ. Contam. Toxicol. 53, 120–126. Langeland, K.A., Laroche, F.B., 1994. Persistence of bensulfuron-methyl and control of Hydrilla in shallow ponds. J. Aquat. Plant Manage. 32, 12–14.
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Larsen, D.P., DeNoyelles, F., Stay, F., Shiroyama, T., 1986. Comparisons of single species, microcosm and experimental pond responses to atrazine exposure. Environ. Toxicol. Chem. 5, 179–190. Wei, L., Hongxia, Y., Yue, S., Jianfang, F., Liansheng, W., 1998. The effects of three sulfonylurea herbicides and their degradation products on the green algae Chlorella pyrenoidosa. Chemosphere 37 (4), 747–751. Newman, M.C., 1995. Quantitative Methods in Aquatic Ecotoxicology. Lewis Publishers, London. € rdog, V., Kuivasniemi, K., 1989. Studies on the effect of cell O division-inhibiting herbicides on unialgal and mixed algal cultures. Int. Revue Ges. Hydrobiol. 74, 221–226. Pimentel, D., McLaughlin, L., Zepp, A., Lakitan, B., Kraus, T., Kleinman, P., Vancini, F., Roach, W.J., Graap, E., Keeton, W.S., Selig, G., 1991. Environmental and economic effects of reducing pesticide use: a substantial reduction in pesticides might increase food costs only slightly. BioSci. -Am.-Inst.-Biol.-Sci. 41, 402–409. Reish, D.L., Oshida, P., 1987. Manual of methods in aquatic environment research. Part 10, Short-term static bioassays. FAO Fisheries Technical Paper 247, Food and Agriculture Organization of the United Nations. Sabater, C., Carrasco, J.M., 1996. Effects of thiobencarb on the growth of three species of phytoplankton. Bull. Environ. Contam. Toxicol. 56, 977–981. Sabater, C., Carrasco, J.M., 1997. Effects of chlorsulfuron on growth of three freshwater species of phytoplankton. Bull. Environ. Contam. Toxicol. 58, 807–813. Sabater, C., Carrasco, J.M., 1998. Effects of molinate on growth of three freshwater species of phytoplankton. Bull. Environ. Contam. Toxicol. 61, 534–540. Sabater, C., Carrasco, J.M., 2001a. Effects of pyridaphenthion on growth of five freshwater species of phytoplankton: a laboratory study. Chemosphere 44 (8), 1775–1781. Sabater, C., Carrasco, J.M., 2001b. Effects of organophosphorus insecticide fenitrothion on growth in five freshwater species of phytoplankton. Environ. Toxicol. Wat. Qual. 16, 314–320. Sabater, C., Tarazona, C., Carrasco, J.M., 2000. Study of the contamination levels of lake of Albufera (Valencia, Spain). (Unpublished data). SAS, 1988. SAS/STAT User’s Guide: Statistics, Version 5. SAS Institute Inc, Cary, NC. Sloof, W., Canton, J.H., Hermens, J.L.M., 1983. Comparison of the susceptibility of 22 freshwater species to 15 chemical compounds. I. (Semi)acute toxicity tests. Aquat. Toxicol. 4, 113–128. STATGRAPHICS, 1994. Statistical Corp., Version 2.1. STSC Inc., Rockville, Md. Thompson, D.G., MacDonald, L.M., Staznik, B., 1992. Persistence of hexazinone and metsulfuron methyl in a mixed-wood/boreal forest lake. J. Agric. Food Chem. 40, 1444–1449. Tomlin, C. (Ed.), 1997. The Pesticide Manual, 11th ed. The British Crop Protection Council, UK. Vighi, M., Sandroni, D., Sbriscia Fioretti, C., 1996. Modeling herbicide fate in paddy fields. In: Del Re, A.A.M., Capri, E., Evans, S.P., Trevisan, M. (Eds.), The Environmental Fate of Xenobiotics. X Symposium Pesticide Chemistry-Modelling Systems, pp. 449–456.
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Worthing, C.R., Hance, R.J., 1991. The Pesticide Manual, ninth ed. The British Crop Protection Council, Survey, UK. Consuelo Sabater Marco received his Doctor of Philosophy degree in Biology at the Polytechnic University of Valencia in 1994. He was a Professor in the Department of Biotechnology at the Polytechnic University of Valencia since 1994. He was a Visiting Fellow at the Agricultural Chemistry Institute (Perugia) and at the Department of Environmental Biology (Siena). Andres Cuesta Martinez received a degree in Agronomic Engineering at the Polytechnic University of Valencia, Spain in 2000. For two years he worked at the Pesticide Laboratory of
Biotechnology Department of the Polytechnic University of Valencia about environmental and aquatic toxicology concerns. Rosa Carrasco De Fez received a Doctor of Philosophy degree in Chemistry at the Polytechnic University of Valencia in Spain in 1999. For five years she worked at the Pesticide Laboratory of Biotechnology Department of the Polytechnic University of Valencia about pesticides photodecomposition and biodegradation concerns. She has also cooperated in several studies about pesticides toxicity to algae. At present, she is working at ‘‘Laboratoire de Chimie Bioorganique et Bioinorganique’’ at the Paris-Sud XI University, Orsay.
Effects of bensulfuron-methyl and cinosulfuron on growth of four freshwater species of phytoplankton C. Sabater *, A. Cuesta, R. Carrasco Pesticide Laboratory, Department of Biotechnology, E.T.S.I.A., Polytechnic University of Valencia, Camino de Vera 14, 46022 Valencia, Spain Received 26 April 2001; accepted 17 August 2001
Abstract The acute toxicity of sulfonylurea herbicides bensulfuron-methyl and cinosulfuron was tested on the five species of freshwater phytoplankton: Scenedesmus acutus, Scenedesmus subspicatus, Chlorella vulgaris and Chlorella saccharophila. Herbicide concentrations eliciting a 50% growth reduction over 96 h ðEC50 Þ ranged from 8 to 104 mg/l for cinosulfuron and from 0.015 to 6.2 mg/l for bensulfuron-methyl. The pesticides bensulfuron-methyl, atrazine and benthiocarb were more toxic than cinosulfuron, chlorsulfuron, molinate, fenitrothion and pyridaphenthion in a toxicity study with the same algal species. The transformation of effective concentrations of bensulfuron-methyl and cinosulfuron and other pesticides, obtained from toxicity measurements, into percent of the saturation level in water is used as a first evaluation of potential hazard to aquatic systems. The herbicides cinosulfuron, methyl-bensulfuron, atrazine and chlorsulfuron were more dangerous than the herbicides benthiocarb and molinate and than the insecticides fenitrothion and pyridaphenthion, in a study of hazard evaluation. The two species of Chlorella were more tolerant to both herbicides than the two species of Scenedesmus. A potential environmental hazard of sulfonylurea herbicides to aquatic systems has to be expected even at low environmental concentrations. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Toxicity; Herbicide; Bensulfuron-methyl; Cinosulfuron; Algae
1. Introduction A wide range of pesticides are used to protect agricultural crops. Although the instructions for use of these chemicals aim at minimizing the risk of contamination of aquatic environments, residuals of pesticides can be detected in water courses draining agricultural areas (K€ allqvist and Romstad, 1994). In this way, the sulfonylurea herbicides such as bensulfuron-methyl and cinosulfuron are characterized by broad-spectrum weed control at very low use rates (2–75 g/ha), good crop
*
Corresponding author. Tel.: +34-9-638-77428; fax: +34-9638-77429. E-mail address: [email protected] (C. Sabater).
selectivity, very low acute and chronic animal toxicity and no propensity to bioaccumulate in non-target organisms. All these characteristics make the sulfonylurea herbicides ideal candidates for replacing some of the older herbicides in an effort to reduce the quantity of chemicals used (Pimentel et al., 1991) and to eliminate herbicides with relatively high animal toxicity (Fletcher et al., 1993). This class of herbicides acts through inhibition of acetolactate synthase (specific to plants and microorganisms) and thereby blocking the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. This inhibition leads to the rapid cessation of plant cell division and growth (Brown, 1990). Bensulfuron-methyl [methyl 2-[[[[[(4,6-dimethoxy-2pyrimidinyl) amino] carbonyl] amino] sulfonyl] methyl] benzoate] controls many problem broadleaf weeds and
0045-6535/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 1 7 9 - 5
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sedges in continuously flooded rice, at 30–100 g/ha. In combination with thiocarbamate grass herbicides, bensulfuron-methyl provides total season-long weed control with good crop safety (Beyer et al., 1988; Tomlin, 1997). Cinosulfuron [N-[[(4,6-dimethoxy-1,3,5-triazin-2-yl) amino] carbonyl]-2-(2-methoxyethoxy) benzenesulfonamide] is applied post-emergence to control many weeds in transplanted, direct seeded, wet-sown and watersown, and dry-sown flooded rice crops, at 20–80 g/ha (Tomlin, 1997). Because these herbicides are typically applied to rice fields, contamination of nearby aquatic systems may occur through a variety of mechanisms, including overspray, drift, surface runoff and leaching (Thompson et al., 1992). It is important to assess the adverse impacts these chemicals may have on non-target organisms in aquatic ecosystems. Wetland contamination could result in a die-off of most algal species present, causing complete disappearance of this food source. Alternatively, certain species or groups of algae could be selectively inhibited. Algae and cyanobacteria are known to be comparatively sensitive to many chemicals, and the inclusion of these organisms in test batteries has been shown to improve the capacity of the battery to predict the most sensitive ecosystem responses (Sloof et al., 1983). Furthermore, the importance of these organisms as dominating primary producers in most aquatic ecosystems speaks for their use in test batteries for environmental hazard assessment. An examination of published phytoplankton toxicity data demonstrates that wide differences in sensitivity can occur across phylogenetic groups of phytoplankton (Blanck et al., 1984; K€allqvist and Romstad, 1994; Fairchild et al., 1997). Much research concerning the toxicity of sulfonylurea herbicides on weeds and crops has been reported (Beyer et al., 1988; Fletcher et al., 1993; Dastgheib et al., 1995); there are few reports about their effects on aquatic organisms. The objective of this work was to investigate the effective concentrations of bensulfuron-methyl and cinosulfuron that caused 50% inhibition on growth, in pure cultures, of two wild species of phytoplankton, representatives of Mediterranean wetlands, isolated from Lake Albufera (Valencia, Spain) and two laboratory strains.
2. Materials and methods The chlorophyceae Scenedesmus acutus (Meyens), Scenedesmus subspicatus CCAP 276/22, Chlorella vulgaris Beijerinck and Chlorella saccharophila (Kr€ uger) Migula were selected for the toxicity tests. S. acutus and C. saccharophila were isolated from samples collected at the Albufera lake in Valencia (Spain). S. subspicatus and C. vulgaris were kindly supplied by the Institute of
Freshwater Ecology (Ambleside, UK) and by the Area of Environmental Toxicology (CISA-INIA, Spain), respectively. These four chlorophyceae were grown in a medium recommended by the ASTM (1993). The stock cultures were maintained in a liquid medium at a temperature of 22 2 °C and a light intensity of 1100 lux on a 12-h light-dark cycle. Bensulfuron-methyl (99.4%, analytical standard) and cinosulfuron (98%, analytical standard) were obtained from DuPont Iberica and Ciba-Geigy (Valencia, Spain). Bensulfuron-methyl (pure) is a solid with molar mass 396.4 and with melting point 185–188 °C and vapor pressure 17 nPa (25 °C); its Kow is 155 (pH 5) and 4.1 (pH 7) and its pKa 5.2; its solubility (25 °C) in water is 2.9 mg/l at pH 5 and 120 mg/l at pH 7; its solubility (20 °C) in acetonae, acetonitrile, dichloromethane and hexane is 1.38, 5.38, 11.7 and 0.31 mg/l, respectively. Cinosulfuron (pure) is a colorless crystalline powder with a molar mass of 413.4; water solubility of cinosulfuron (20 °C) is 18 mg/l (pH 2.5), 82 mg/l (pH 5) and 3.7 g/l (pH 7); its solubility (20 °C) in dichloromethane and dimethyl sulfoxide is 95 and 320 g/kg, respectively, and is slightly soluble in common organic solvents; its melting point and vapor pressure (20 °C) is 144.6 °C and 100 pPa, respectively; its Kow at (25 °C) is 2.04 (pH 2.1) and 0.63 (pH 7) and its pKa 4.72 (Worthing and Hance, 1991; Tomlin, 1997). The inhibition test was conducted in accordance with the ASTM (1993) protocol. The organisms were exposed to various concentrations of bensulfuron-methyl and cinosulfuron for 96 h. Growth of cultures was measured by the turbidity at 750 nm wavelength using a spectro€ rdog and Kuivasniphotometer (Beckman DUâ -70) (O emi, 1989) at 24, 48, 72 and 96 h after the start of the test. After 96 h, growth rates were quantitatively determined using the exponential model y ¼ eaþlx [where y ¼ the population estimate, x ¼ time, a ¼ integration constant and l ¼ slope or growth rate]. ANOVA and Student Newman–Keuls multiple range test were employed to determine if treatments were significantly different from each other (Reish and Oshida, 1987). Results were deemed significantly different at the level P 6 0:05. EC10 (0–96 h) and EC50 (0–96 h) values with 95% confidence limits were estimated by the linear regression of probit of percentage growth on the log dose of bensulfuron-methyl and cinosulfuron (Newman, 1995). In this study, EC10 and EC50 are the concentrations of herbicides, derived by the method of calculation ‘‘comparison of areas under growth curves’’, which results in a 10% and a 50% growth reduction relative to the control values, at 96 h, respectively (ASTM, 1993). All statistical analyses were performed using the commercial software packages (STATGRAPHICS, 1994) (STSC, Rockville, Md.) and SAS (1988) (SAS Institute, Cary). Further details of test protocols are provided in Sabater and Carrasco (1996).
C. Sabater et al. / Chemosphere 46 (2002) 953–960
At the beginning and at the end of the assays, herbicide concentrations in test solutions were determined by HPLC; the results were analyzed statistically and used for calculating the toxicities of bensulfuron-methyl and cinosulfuron. For the extraction and determination of herbicides the test culture samples (25 ml) were filtered, acidified with HCl 1 N and extracted with three 20 ml portions of dichloromethane by shaking in a separatory funnel. The combined hexane extracts were filtered through NaSO4 to remove residual water and evaporated to dryness and the residue was dissolved in 5–10 ml of acetonitrile/NH3 0.024 M (30/70, v/v) and analyzed by HPLC with absorbance detection at 220 nm. The column was a 15 cm Shodex DS-613 RS-pak and the mobile isocratic phase was acetonitrile/aqueous phosphate (103 M) acidified with phosphoric acid (pH 4.6), (60:40, v/v) (Galletti et al., 1995; Carrasco, 1999). Approximated retention time for bensulfuron-methyl is 5:71 0:17 min and for cinosulfuron is 3:72 0:04 min. The recovery of bensulfuron-methyl and cinosulfuron was in the range between 93 2% and 90 5%, respectively.
3. Results and discussion Experimental bensulfuron-methyl and cinosulfuron exposure concentrations measured by GC (no nominal values) are shown (Tables 1 and 2). The concentrations of bensulfuron-methyl that caused significant effects ðP < 0:05Þ on the algal growth with respect to control values ranged from 5.0 to 18.9 mg/l for C. saccharophila, from 0.21 to 15.5 mg/l for C. vulgaris, from 0.0083 to 0.32 mg/l for S. acutus and from 0.035 to 3.6 mg/l for S. subspicatus. The concentrations of cinosulfuron that caused significant effects ðP < 0:05Þ on the algal growth were markedly higher, between 41.5 and 358.0 mg/l for C. saccharophila, between 62.3 and 355.5 mg/l for C.
955
vulgaris, between 3.6 and 98.8 mg/l for S. acutus and between 11.6 and 150.7 mg/l for S. subspicatus. Growth of the four species of phytoplankton under different exposures of bensulfuron-methyl and cinosulfuron to that of controls was compared (Tables 1 and 2). A broad range of sensitivity within species was observed for both herbicides. Reduction in growth rates was observed with an increase of bensulfuron-methyl and cinosulfuron concentrations. At 5.0, 0.21, 0.0083 and 0.035 mg/l of bensulfuron-methyl the growth of C. saccharophila, C. vulgaris, S. acutus and S. subspicatus was significantly inhibited. However, doses of cinosulfuron greater than 41.5 mg/l for C. saccharophila, 62.3 mg/l for C. vulgaris, 3.6 mg/l for S. acutus and 11.6 mg/l for S. subspicatus were necessary for producing similar bensulfuron-methyl effects on the growth of these species. In contrast to Chlorella species, whose growth was strongly inhibited by concentrations of bensulfuron-methyl of 15.5–18.9 mg/l (lethal concentrations), Scenedesmus species were highly sensitive at lower concentrations of this herbicide, 0.32–3.6 mg/l. No inhibition of the growth of Chlorella species was observed at 26.1 and 36.8 mg/l of cinosulfuron, respectively. Conversely, strong inhibition of S. acutus growth was observed at 35.9 mg/l of this herbicide. Based on their 96-h EC10 and EC50 values, the four species of algae responded very differently to bensulfuron-methyl and cinosulfuron. Scenedesmus species were more sensitive to both herbicides than were Chlorella species. S. acutus and S. subspicatus both proved to be very sensitive to bensulfuron-methyl, with 10% and 50% inhibition of growth at bensulfuron-methyl concentrations of 0.0052–0.0095 and 0.015–0.034 mg/l, respectively. The 96 h EC10 and EC50 values of cinosulfuron for algae assayed were markedly higher than were for bensulfuron-methyl, ranging from 1.8 to 36 mg/l for Scenedesmus species and from 30 to 104 mg/l for Chlorella species. The toxicities of bensulfuron-methyl and
Table 1 Average specific growth rates (la ) of the four species of phytoplankton treated with bensulfuron-methyl C. saccharophila
C. vulgaris
S. acutus
S. subspicatus
mg/l
l
R2
mg/l
l
R2
mg/l
l
R2
mg/l
l
R2
0.0 2.3b 5.0 5.9 7.0 18.9c
0.77 0.077 0.075 0.073 0.066 –
0.93 0.93 0.96 0.98 0.99 –
0.0 0.010b 0.21 1.6 5.7 15.5c
0.063 0.063 0.059 0.056 0.047 –
0.98 0.97 0.97 0.98 0.98 –
0.0 0.0017b 0.0083 0.017 0.034 0.32c
0.059 0.059 0.057 0.053 0.039 –
0.97 0.97 0.97 0.98 0.92 –
0.0 0.0022b 0.035 0.053 0.15 3.6c
0.059 0.059 0.056 0.049 0.044 –
0.92 0.92 0.99 0.99 0.99 –
R2 : Correlation coefficient. a Growth rates (l) were determined using the exponential model, y ¼ eaþlx [y ¼ the population estimate, x ¼ time, a ¼ integration constant and l ¼ slope or growth rate]. b Highest concentrations tested without significant effects ðP < 0:05Þ on the algal growth relative to control values. c Lethal concentrations.
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Table 2 Average specific growth rates (la ) of the four species of phytoplankton treated with cinosulfuron C. saccharophila
C. vulgaris 2
S. acutus 2
S. subspicatus 2
mg/l
l
R
mg/l
l
R
mg/l
l
R
mg/l
l
R2
0.0 26.1b 41.5 65.4 112.9 358.0c
0.077 0.077 0.075 0.072 0.067 –
0.96 0.94 0.95 0.97 0.98 –
0.0 36.8b 62.3 93.9 112.8 355.0c
0.057 0.057 0.056 0.051 0.039 –
0.99 0.99 0.99 0.99 0.98 –
0.0 0.52b 3.6 15.0 35.9 98.8c
0.059 0.059 0.056 0.051 0.036 –
0.95 0.96 0.98 0.99 0.97 –
0.0 2.5b 11.6 36.4 78.0 150.7c
0.060 0.060 0.059 0.055 0.047 –
0.92 0.91 0.97 0.99 0.99 –
R2 : Correlation coefficient. a Growth rates (l) were determined using the exponential model, y ¼ eaþlx [y ¼ the population estimate, x ¼ time, a ¼ integration constant and l ¼ slope or growth rate]. b Highest concentrations tested without significant effects (P < 0:05) on the algal growth relative to control values. c Lethal concentrations.
Table 3 Algal EC10 and EC50 (0–96 h) values of bensulfuron-methyl and cinosulfuron with 95% confidence limits for the four species Test species
Herbicides Cinosulfuron (mg/l)
C. saccharophila C. vulgaris S. acutus S. subspicatus
Bensulfuron-methyl (mg/l)
EC10
EC50
EC10
EC50
30 (28–34) 38 (31–46) 1.8 (0.9–2.3) 10.2 (9.5–13.0)
104 (89–128) 96 (94–98) 8 (4–13) 36 (32–40)
2.7 (1.4–3.4) 0.40 (0.36–0.44) 0.0052 (0.0041–0.0068) 0.0095 (0.0088– 0.0130)
6.2 (6.0–6.3) 2.2 (1.8–2.6) 0.015 (0.014–0.017) 0.034 (0.031–0.037)
cinosulfuron to our species of phytoplankton and toxicity data published for several species of phytoplankton with another sulfonylurea herbicides have shown that the variation in sensitivity may be considerable. Blanck et al. (1988) showed that the range in susceptibility among species to chemicals extended over more than three orders of magnitude. Ninty-six-hour EC50 values of bensulfuron-methyl obtained in our study for C. saccharophila and C. vulgaris have been 2.2 and 6.2 mg/l, respectively. These values are higher than the 96-h EC50 of 0.17 mg/l found by Liping Wei et al. (1998) for growth inhibition in Chlorella pyrenoidosa exposed to bensulfuron-methyl. This value of 0.17 mg/l is, at the same time, greater than bensulfuron-methyl 96-h EC50 of 0.015 mg/l for S. acutus and than bensulfuron-methyl 96-h EC50 of 0.034 mg/l for S. subspicatus (Table 3). Metsulfuron-methyl, chlorsulfuron, triasulfuron and tribenuron-methyl affected growth and reproduction of Chlorella fusca at concentrations between 0.04 and 4 mg/l (Fahl et al., 1995) and Hartnett et al. (1987) observed growth inhibition of Chlamydomonas reinhardtii on agar plates by sulfometuron-methyl at 0.36 mg/l or greater. EC50 values for the effect of chlorsulfuron on the growth rate of several phytoplankton species ranged from 0.15 to 60 mg/l, a total range factor of 400 (Dyer et al.,
1982; K€ allqvist and Romstad, 1994; Carrasco and Sabater, 1997). The macrophyte Lemna minor was highly sensitive to chlorsulfuron (0.7 lg/l; 96-h EC50 ) and metsulfuron ð0:4 lg=l; 96-h EC50 Þ, whereas the green algae Selenastrum capricornutum was 100-fold sensitive (chlorsulfuron, 135 lg=l 96-h EC50 ; metsulfuron, 190 lg=l 96-h EC50 ) (Fairchild et al., 1997). Although a high degree of variability was observed by these authors among the algal species studied, the majority of sulfonylureas EC50 values were in the range from 0.08 to 54 mg/l. However, in our study we found an EC50 for bensulfuron-methyl in Scenedesmus species of 0.015–0.034 mg/l and an EC50 for cinosulfuron in Chlorella species of 96–104 mg/l, which were out of the mentioned range found in the bibliography. The significance of the large interspecies variation in sensitivity among phytoplankton species is not obvious. Exposure of a natural phytoplankton community to a toxic chemical will tend to extinguish the most sensitive species. Because of the generally high reproduction potential of phytoplankton, other species will rapidly replace those that disappear and the standing crop and level of primary production may remain merely unchanged (K€ allqvist and Romstad, 1994). The new community will have a higher tolerance to the specific pollutant, as has been shown experimentally by Blanck
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et al. (1988), who introduced the term ‘‘pollution induced community tolerance’’ (PICT) for this phenomenon. Environmental concentrations of bensulfuron-methyl and cinosulfuron of 0.007–0.025 and 0.003–0.03 mg/l, respectively, have been reported in flooded paddy fields (Allievi, 1996). Concentrations of bensulfuron-methyl are much lower than toxicity values of 0.40 and 6.2 mg/l obtained for Chlorella species and are near to the EC50 of 0.034 mg/l for S. subspicatus but exceeded EC10 values for Scenedesmus species and EC50 value for S. acutus (Table 3), as determined in this study. With regard to ecotoxicological risk assessment for the development of Scenedesmus species under environmental conditions the NOEC and EC10 values (0.0017–0.0095 mg/l) were in the same range as the concentration levels of bensulfuronmethyl found in flooded paddy fields (Allievi, 1996) and as the concentration levels of 0.0003–0.0084 mg/l found by Sabater et al. (2000) at different points in waters from Lake Albufera (Valencia, Spain) in spring and summer of 2000; so the use of bensulfuron-methyl may cause harmful effects on the long-term development of S. acutus and S. subspicatus natural populations. The loss of a few, particularly sensitive, phytoplankton species from a community containing hundreds of species may not be considered significant, as long as the function of the community remains unchanged. However, this may be one of the most sensitive reactions of an ecosystem to a pollution stress, and thus be an important indication of the potential of ecologically harmful effects (K€allqvist and Romstad, 1994). A potential environmental hazard of sulfonylurea herbicides to aquatic systems has to be expected even at
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low environmental concentrations but the ecological fate of bensulfuron-methyl may be changed substantially under several conditions. In rice paddy water, microbial degradation is an important factor in the breakdown of bensulfuron-methyl (Langeland and Laroche, 1994). Chemical hydrolysis was often considered as secondary, but its occurrence must not be completely neglected. In an evaluation of the risk for the receiving surface waters for River Po system (Vighi et al., 1996) the disappearance of bensulfuron-methyl was very rapid, among the water applied pesticides in paddy fields, and their risk was considered absent or negligible throught year with the exception of the month of May. Liping Wei et al. (1998) found that bensulfuron-methyl can be degraded and so would decrease fastly their concentrations into the aquatic media which lead to a rapid decrease in toxicity to the green algae. Toxicity data were further examined for fenitrothion and other pesticides with same algal species, by normalizing EC50 values to water solubility and molar mass by using the percent saturation approach (Gaggi et al., 1995). In this way a first-step evaluation of the hazard to aquatic systems is produced, indicating how far the effective concentration is from the maximum possible in water (Table 4). The combined use of the effective concentrations with percent saturation leads to two results: a classical toxicity ranking and a preliminary hazard classification for aquatic contaminants. Three response groups were observed in Chlorella species, the first about 0.0063–0.25% saturation for clorsulfuron, cinosulfuron, bensulfuron-methyl and atrazine, the second about 1.6–4.3% saturation for molinate and
Table 4 Hazard evaluationa of several compounds to freshwater algae Pesticide Atrazine Benthiocarb Cinosulfuron Chlorsulfuron Fenitrothion Bensulfuron-methyl Molinate Pyridaphenthion a
Test species C. saccharophila
C. vulgaris
S. acutus
S. subspicatus
0.085b 13.4e 0.25 0.0063g 27.1h 0.13 1.6i 18.9j
0.031c 8.4f 0.23 – 56.6h 0.046 4.3i 8.9j
0.0016b 0.057e 0.019 0.00003g 4.0h 0.00032 0.099i 1.5j
0.021d 0.54f 0.086 – 6.2h 0.00072 0.072i 1.3j
EC50 values were transformed into percent saturation, referred to the solubility in water of the chemical as subcooled liquid (Gaggi et al., 1995). b Data from Carrasco and Sabater (1997). c Data from Larsen et al. (1986). d Data from Kirby and Sheahan (1994). e Data from Sabater and Carrasco (1996). f Data from Sabater et al. (2000). g Data from Sabater and Carrasco (1997). h Data from Sabater and Carrasco (2001b). i Data from Sabater and Carrasco (1998). j Data from Sabater and Carrasco (2001a).
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the third about 8.4–56.6% saturation for fenitrothion, pyridaphenthion and benthiocarb. In Scenedesmus species were also observed three response groups, the first about 0.00003–0.0016% saturation for clorsulfuron, bensulfuron-methyl and atrazine, the second about 0.019–0.54% saturation for molinate, benthiocarb and cinosulfuron and the third about 1.3–6.2% saturation for fenitrothion and pyridaphenthion. A comparison of the relative sensitivity of Chlorella and Scenedesmus species to the seven pesticides is presented in Figs. 1–4. Data points that fall on the line (i.e., slope ¼ 1) indicate equivalent sensitivity of the two species to a given chemical. Data that fall to the right of the line indicate that C. vulgaris or S. subspicatus are more sensitive; alternatively, data that fall to the left of the line indicate that C. saccharophila or S. acutus are more sensitive. The collective data indicate that C. vulgaris and S. acutus were more sensitive to pesticides (Figs. 1–4). A large degree of variation in sensitivity among species to both sulfonylureas was obvious in our study, with the growth of same species being completely unaffected by concentrations that were phytotoxic to other species. The two species of Chlorella were considerably more tolerant than two species of Scenedesmus. S. acutus was the most sensitive alga and C. saccharophila the least
Fig. 3. Hazard evaluation of seven pesticides on S. acutus and S. subspicatus.
Fig. 4. Toxicity of seven pesticides on S. acutus and S. subspicatus.
sensitive. The pesticides methyl-bensulfuron, atrazine and benthiocarb were more toxic than cinosulfuron, chlorsulfuron, molinate, fenitrothion and pyridaphenthion in a toxicity study with the same algal species but the herbicides cinosulfuron, bensulfuron-methyl, atrazine and chlorsulfuron were more dangerous than the herbicides benthiocarb and molinate and than the insecticides fenitrothion and pyridaphenthion, in a study of the hazard evaluation. Fig. 1. Hazard evaluation of seven pesticides on C. vulgaris and C. saccharophila.
Acknowledgements This work was supported by a grant from ‘‘Comisi on Interministerial de Ciencia y Tecnologia’’ (1FD97-0963C03-03) and from ‘‘Generalitat Valenciana’’ (GV-DAG-01-154-96).
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Fig. 2. Toxicity of seven pesticides on C. vulgaris and C. saccharophila.
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Worthing, C.R., Hance, R.J., 1991. The Pesticide Manual, ninth ed. The British Crop Protection Council, Survey, UK. Consuelo Sabater Marco received his Doctor of Philosophy degree in Biology at the Polytechnic University of Valencia in 1994. He was a Professor in the Department of Biotechnology at the Polytechnic University of Valencia since 1994. He was a Visiting Fellow at the Agricultural Chemistry Institute (Perugia) and at the Department of Environmental Biology (Siena). Andres Cuesta Martinez received a degree in Agronomic Engineering at the Polytechnic University of Valencia, Spain in 2000. For two years he worked at the Pesticide Laboratory of
Biotechnology Department of the Polytechnic University of Valencia about environmental and aquatic toxicology concerns. Rosa Carrasco De Fez received a Doctor of Philosophy degree in Chemistry at the Polytechnic University of Valencia in Spain in 1999. For five years she worked at the Pesticide Laboratory of Biotechnology Department of the Polytechnic University of Valencia about pesticides photodecomposition and biodegradation concerns. She has also cooperated in several studies about pesticides toxicity to algae. At present, she is working at ‘‘Laboratoire de Chimie Bioorganique et Bioinorganique’’ at the Paris-Sud XI University, Orsay.