Fenthion loss in 0.45 μm GF/F-filtered seawater during 96 h of illumination of cool-white fluorescent lights (4300 Lux or 12.9 W m−2) and at 20.0 ± 0.3°C.
This chapter provides the results of a laboratory ecotoxicological study conducted to assess the acute toxicity of the organophosphorus pesticide fenthion toward the marine microalgal species Tetraselmis suecica. Bioassays were performed, and algal densities and chlorophyll pigments fractions were measured in the exponential phase after 96 h of exposure to fenthion. Two quantitative structure activity relationships (QSARs) were used to estimate the toxicity of 13 primary metabolites and degradation products of fenthion toward the selected organism; the first was based on the use of the n-octanol/water partition coefficient, whereas the second was based on the solubility of the compound in water. Results revealed that fenthion can have marked effects on the growth and photosynthesis of the target primary producers of marine ecosystems T. suecica. The parent pesticide toxicant was found not toxic to the tested algal species up to 1.00 mg L−1, while higher treatment concentrations not only affected algal densities and significantly decreased specific growth rate values (μ) (p < 0.05) but also decreased the contents of photosynthetic pigments. The comparison between the observed and the predicted toxicity values of the parent compound fenthion indicated that the predictive capability of the QSARs applied can be considered highly satisfactory. Consequently, both QSAR models were used for the prediction of toxicity data of fenthion’s principal metabolites and degradation products.
- Tetraselmis suecica
- toxicity test
- pigment biomarker
In Greece, only one formulation of fenthion was registered by Bayer CropScience Hellas, with the trade name LEBAYCID 50% EC (containing 50% w/v fenthion as the active ingredient), which is classified as dangerous for humans, but is not classified for aquatic organisms . This insecticide is extremely effective in controlling the major insects infecting olives, such as the olive fruit fly
The available information on the production and use of pesticides in general and hence of organophosphates as well is limited, fragmentary and in some cases unreliable . On the basis of the limited information received from the Mediterranean countries, fenthion was one of the important compounds used during the 1980s and 1990s among other organophosphorus pesticides . According to data provided by the Greek Ministry of Rural Development and Food, it appears that the quantities of fenthion that were used for agricultural purposes during the years 1983, 1984, 1985, 1986, 1987, 1988, and 1989 in Greece were 216,892; 409,139; 24,359; 197,843; 87,787; 160,433; and 213,514 tons of active ingredient, respectively .
Since June 2007, fenthion is no longer approved by the Greek Ministry of Rural Development and Food because of an excess number of poisoning-related events and ecotoxicology effects on nontarget organisms (Greek Ministry Decision, Register Number 122914–27/4/2005, 2005), apart from its 120 days of exceptional authorization (from May 1, 2009 to August 31, 2009) in accordance with Art. 8(4) of Directive 91/414/EEC for the treatment of olive trees against
Although fenthion was developed as a safe pesticide because it is not easily converted to the possibly highly toxic oxon derivative (fenthion oxon) in mammalian species, however according to relative literature, many of its metabolites were detected in various plants, animals, and environmental matrices [4, 5, 6, 7]. Kitamura et al. demonstrated that the in vivo metabolism of fenthion in fish leads to the formation of two metabolites, fenthion sulfoxide and fenthion oxon , while other studies proved that fenthion and its oxidation products were accumulated in fish . Oxidation products of fenthion, including fenthion oxon, were also detected in house mosquitoes exposed to fenthion . It has also been reported that fenthion was converted to fenthion oxon in the aqueous environmental bodies . On the contrary, the toxicity and the metabolism of this organophosphorus insecticide have not been extensively studied in aquatic microspecies, such as microalgae.
Microalgae are important inhabitants of aquatic ecosystems, where they fulfill critical roles in primary productivity, nutrient cycling, and decomposition. Detrimental effects of pesticides on algae may have subsequent impacts on higher trophic levels . It has been well established that changes in the macromolecular composition of phytoplankton species or shifts in community composition can affect the growth rate of zooplankton grazers . Unquestionably, aquatic environments receive direct and indirect pesticide inputs, inevitably exposing microorganisms to pesticides. Millions of pounds of active pesticide ingredients are applied in coastal watersheds each year, and in addition, pesticides may affect marine inhabitants via spills, runoff, and drift .
Toxicity data involving ecotoxicology of fenthion toward nontarget microorganisms are limited. Most studies have focused on microbial degradation and biotransformation of fenthion rather than impacts on natural microbial populations and communities. Furthermore, studies of fenthion effects on soil microbes are far more common than studies of toxicology assessments in aquatic environments. Published data regarding marine or estuarine microorganisms are even scarcer .
The aims of the present survey were (i) to assess the acute toxicity of the organophosphorus insecticide fenthion toward nontarget aquatic microorganisms, such as marine algae, (ii) to investigate the possibility of using the parameter of chlorophyll pigments as biomarkers of exposure to fenthion, (iii) to compare the observed and predicted endpoint toxicity data and evaluate the predictive capability of two QSARs based on physicochemical properties of target organic toxicant (n-octanol/water partition coefficient and water solubility), and (iv) to predict the toxicity of 13 principal metabolites and degradation products of fenthion toward the selected marine microalgae.
2. Materials and methods
2.1. Organism and culture conditions
Unialgal cultures of the species were maintained in liquid
One hundred milliliters of inoculated growth medium
2.2. Test chemicals, reagents, and standards
The tested compound fenthion was an analytical grade (purity >99.5%), obtained from Dr. Ehrenstorfer-Schäfers (Augsburg, Germany) and used without further purification. Pure fenthion is a colorless, almost odorless liquid, while technical product of fenthion (95–98% pure) is a brown oily liquid with a weak garlic odor. Data for other physiochemical properties of fenthion, taken from reference , include melting point, 7°C; boiling point, 87°C at 0.01 mmHg; vapor pressure, 1.4 mPa at 25°C; water solubility, 55 mg L−1 (at 20°C and pH = 7); log Kow, 4.84; and Mr, 278.34. Figure 1 shows the chemical structure of the target compound.
Due to low water solubility of the tested substance, acetone was used for the preparation of its stock solutions. Hence, acetone was used as the carrier solvent of the compound to the bioassays, since previous experiments proved that this solvent up to a final concentration of 0.5 μL mL−1 in
Pesticide-grade organic solvents such as acetone, hexane, methanol, and dichloromethane were purchased from Pestiscan (Labscan Ltd., Ireland). Organic-free water was prepared with a Milli-Q/Milli-Ro system (Millipore Corp., Bedford, USA). Other chemical reagents and solvents used were of HPLC grade and procured from Merck (Merck, Germany).
2.3. Procedure for the study of the stability of fenthion
The stability of fenthion in seawater was determined under the experimental conditions employed for the incubation of the cultures. Therefore, parallel experiments were performed without algae using all six test concentrations that were chosen for the toxicity treatments (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg L−1) and prepared in 0.45 μm GF/F-filtered natural seawater. Triplicate samples were prepared in 250 mL borosilicate flasks, and each contained 100 mL of pesticide solution. At various time intervals (0, 24, 48, 72, and 96 h, as for the toxicity tests), 5 mL aliquots of the aqueous reaction solutions were withdrawn for analysis.
Fenthion concentrations were quantitatively confirmed by gas chromatography analysis after liquid–liquid extraction of the fortified aqueous samples with hexane (2 × 5 mL). Organic extracts were dried over anhydrous sodium sulfate, and 1 μL of the extracts was injected into gas chromatographic system (Hewlett-Packard 5890) equipped with a nitrogen-phosphorus detector (GC-NPD). A 30 m × 0.32 mm i.d. × 0.25 film thickness fused silica-bonded phase capillary column (MDN-5, Supelco, USA) was used for the chromatographic separation of target analyte and oven temperature programmed at 150°C for 3 min; increased from 150–170°C at 20°C min−1; then increased from 170–190°C at 2°C min−1; after that increased from 190–250°C at 15°C min−1; and was held to 250°C for 15 min. Helium was used as the carrier gas at constant flow of 1.2 mL min−1 during GC analysis. Injection technique was on column. Detector’s temperature was 280°C, while hydrogen and air were used as NPD’s airs with flows of 3.5 and 110 mL min−1, respectively.
2.4. Acute toxicity test and pesticide treatment
Bioassays were performed according to the OECD Guideline 201 for testing the effects of chemicals on alga growth inhibition test , with some modifications. Cells in the exponential phase of growth were collected from stock cultures (called as pre-cultures and incubated under the previously mentioned conditions) and for this reason used as the inoculum. The initial algal density in each one of the experimental treatments was of 1 × 105 cells mL−1 .
The experimental design and test conditions were identical for all replicates performed. Each chemical bioassay included the below-described treatments: a control (C) containing no pesticide; a control containing acetone as carrier solvent of the organic toxicants, in concentration 0.05% (C + A); and various toxicant exposure concentrations of fenthion (in mg L−1), following the results of preliminary range-finding experiments conducted for the tested compound previously . Algae were exposed to the concentration series of 0.50, 1.00, 1.50, 2.00, 2.50, and 3.00 mg L−1 of fenthion, respectively. Each treatment contained three replicate flasks. The environmental conditions during the experiments were the same as the growth conditions stated in paragraph 2.1.
Cell densities were assessed daily (after 0 h, 24 h, 72 h, and 96 h of incubation) by microscope counting using an improved Neubauer hemocytometer. Lugol solution was added to the samples (ratio of lugol/culture media, 1/10 v/v) to prevent the natural movement of
The contents of acetone-soluble chlorophyll pigments, chlorophyll-a (Chla), chlorophyll-b (Chlb), and chlorophyll-c (Chlc), contained in 10 mL of culture medium at the end of incubation (96 h), were determined according to the spectrophotometric method described in detail by Strickland and Parsons .
2.5. Prediction of toxicity values of primary metabolites and degradation products of fenthion: Quantitative structure activity relationships (QSARs)
Two structure-toxicity relationships have been proposed by Vagi  for the growth inhibition of the marine microalga
Since the experimental determination of log
2.6. Data reliability and statistical analysis
Independent experiments were repeated three times, and each sample (treatment and/or control culture) was repeated three times. Mean values ± standard deviations (SD) are shown in the figures, and tables are presented in this chapter. Data collected were calculated as percentages, and arcsine was transformed (arcsine ) and analyzed using one-way analysis of variance (ANOVA). Variances were considered equal (p > 0.05) based on Kolmogorov–Smirnov test for homogeneity of variance. The highest concentration of toxicant demonstrating no effect as compared to the controls was estimated by Dunnett’s test for statistical significance (p > 0.05) with SPSS software program.
3.1. Fenthion stability
Experimental data of the present study concerning the stability of fenthion in 0.45 μm GF/F-filtered seawater during 96 h of exposure to illumination of cool-white fluorescent lights (4300 Lux or 12.9 W m−2) and at 20.0 ± 0.3°C are summarized in Table 1.
|Treatment concentration (mg L−1)||Remaining pesticide (%) over time|
It must be mentioned that the loss of the target organophosphorus toxicant from solutions over 96 h was similar in all three replicates (data not shown), and the mean values of triplicates are presented. It is obvious that the initial test concentrations (0 h) were between 97.48 and 102.98% of the nominal concentrations, while after 96 h of exposure (under 4300 Lux and at 20 ± 0.3°C), concentration of fenthion in seawater had reduced to between 91.95 and 93.62% of the nominal concentrations. This percentage of loss (less than 10%) is in accordance with the condition set by the OECD for the validity of the test that requires no more than 20% of the test chemical to be lost during the conducted toxicity test .
3.2. Toxicity of fenthion on growth of marine alga
Algae exhibit several responses to toxicants, including growth inhibition and stimulation and morphological and physiological changes . During the performance of present bioassays, the algal cells changed morphologically when treated with fenthion and observed under the optical microscope. Changes in cell shape, color, and size were observed as some cells became darker in color and in other cases were swollen as well. Moreover, many cell divisions were found abnormal because when the material cell divided, the descendant cells remained attached and the daughter cells were not separated. Thus, usual algal aggregations could also be observed. The above phenomenon indicated that fenthion could have been a potential of mutagenic effects on
Cultured in different concentrations of fenthion, the algal growth curves of T. suecica are shown in Figure 2(a–f). The results contained in these charts indicated that cells in fenthion-treated medium grew slower than those in control group.
Furthermore, the mean specific growth rates (
Similar results were found by other authors who tested the influence of the organophosphorus insecticide fenitrothion on
Using the toxicity data contained in Table 2, an estimate of NOEC and LOEC values would be 1.00 and 1.50 mg L−1, respectively, while MATC calculated as the geometric mean between the NOEC and LOEC was estimated to be 1.22 mg L−1. The experimental results of the present study confirmed that fenthion is slightly less toxic toward the target marine microalgae than it was previously reported as the values of NOEC, LOEC, and MATC were reported to be 0.50, 1.00, and 0.70 mg L−1, respectively . The percentage of inhibition data relative to growth in untreated controls (%
Figure 3a shows the concentration-response curve of fenthion to
The linear regression equation that was derived from this linear part of the curve is described by Eq. (5):
Acute toxicity values of EC20 and EC50 (in mg L−1) at 96 h were obtained by the above-described relationship (5), and the calculated data are presented in Table 3.
|EC20 96 h||EC50 96 h|
|(mg L−1)||(mol L−1)||(mg L−1)||(mol L−1)|
|1.04||3.74 × 10−6||1.52||5.46 × 10−6|
EC50 values of target compound estimated in the present work are in accordance with toxicity data reported in the literature for the same toxicant toward other green algal species, such as
3.3. Toxicity of fenthion on chlorophyll pigment production of marine algae
Fenthion belongs to a chemical group of pesticides called organophosphates, which share a common mechanism of toxicity; they all affect the nervous system by inhibiting acetylcholinesterase (AChE). The physiological role of AChE is the cleavage of the neurotransmitter acetylcholine at cholinergic synapses and neuromuscular junctions, thereby terminating the neurotransmitter’s effects on the postsynaptic membrane. The toxicity of insecticidal organophosphates also called the anticholinesterase insecticides (anti-ChEs) is based on their inhibition of AChE, which results in interference with proper neurotransmission. Therefore, fenthion is not expected to be a direct inhibitor of pigment synthesis nor to induce a direct oxidative stress as a consequence of its biochemical mode of action that would destroy chlorophyll pigments. However, pigment content may change in response to the cascade of events following contamination with the pesticide, regardless of its different mode of action [28, 29]. Results of the effect of fenthion on the pigments were expressed either as pigment content of the culture or as percentage inhibition of pigment increase. These results are shown in Table 4 and Figure 4.
|(μg L−1)||(pg cell−1)||(μg L−1)||(pg cell−1)||(μg L−1)||(pg cell−1)||(μg L−1)||(pg cell−1)|
|Control + acetone||1263.114||3.249||540.690||1.391||100.796||0.2593||1904.600||4.899||2.34|
From the collected experimental data, it became obvious that fenthion decreased the contents of photosynthetic pigments (Chla, Chlb, Chlc, and Chltot) and statistically significantly different as compared to the controls occurred in photosynthetic activity of
It was clear that while cell density decreased with increasing exposure treatments of fenthion, values of Chla/Chlb ratio remained stable or increased, suggesting that the biomass of algae was affected by the organophosphorus insecticide much more strongly than the structure of the chlorophyll body. These data were in agreement with those of Li et al., who reported that cypermethrin induced a drastic decrease in the growth and photosynthesis of
Linear correlations between cell density and chlorophyll pigment concentrations of chlorophyll-a, chlorophyll-b, chlorophyll-c, and total chlorophyll were calculated and are described by Eqs. (6)–(9), respectively:
where Chla, Chlb, and Chlc are the concentrations of chlorophyll pigments in culture media, Chltot is the sum of Chla, Chlb, and Chlc (all in μg L−1), and N is the cell number (in cells). The above-described linear correlations resulted in high values of correlation coefficients (R2 > 0.8322), a fact which indicated that the use of chlorophyll measurements to estimate biomass concentration is reliable and validated the possibility of using cell chlorophyll content to assess the state of the cells after 96 h of exposure to fenthion, as previously described by other authors for other cases of bioassays [30, 31]. These results confirmed that the commonly accepted hypothesis of chlorophyll pigment content being proportional to growth rate of microalgal species  applies for toxicity assessment of fenthion on marine phytoplanktonic species such as
Acquired values of chlorophyll content expressed in pg. cell−1 are summarized for each pigment in Table 4. Unfortunately, there is lack of available published information on photosynthetic activity of this species, and the few data are restricted only to chlorophyll-a concentrations . It is observed that when incubated with fenthion concentrations equal or below 1.50 mg L−1, the content of chlorophyll-a/cell of
3.4. Toxicity of the metabolites of fenthion on growth of marine alga
The abiotic and biotic degradation of organophosphorus pesticides has been extensively studied in a large number of studies. Various data concerning the metabolism of several organophosphates in terrestrial and aquatic species, either in vivo or in vitro, are available [4, 5, 6]. After the application of Eqs. (3) and (4) for the prediction of the toxicity of 13 principal metabolites and degradation products of fenthion that have been identified in environmental samples, the predicted EC50 values for
|Compound||Parameters||Predicted EC50 96 h (mg L−1)|
|log ||log |
|Fenthion sulfoxide (I)||2.18||240.00||29.47||0.92|
|Fenthion sulfone (II)||2.34||44.86||24.14||1.09|
|Fenthion oxon (III)||2.30||810.00||25.38||0.83|
|Fenthion oxon sulfoxide (IV)||0.87||2597.00||150.94||0.75|
|Fenthion oxon sulfone (V)||0.91||1773.00||143.59||0.78|
|Demethyl fenthion (VI)||3.07||93.78||9.72||1.01|
|Demethyl fenthion sulfoxide (VII)||1.59||1650.00||61.51||0.78|
|Demethyl fenthion sulfone (VIII)||1.67||590.00||55.67||0.85|
|Demethyl fenthion oxon (IX)||1.81||2540.00||46.75||0.75|
|Demethyl fenthion oxon sulfoxide (X)||0.42||19140.00||264.53||0.65|
|Fenthion phenol (XI)||2.49||1067.00||20.02||0.81|
|Fenthion phenol sulfoxide (XII)||1.19||8533.00||101.28||0.69|
|Fenthion phenol sulfone (XIII)||1.04||3163.00||122.11||0.74|
According to predicted EC50 values of Eq. (3), the parent chemical was more toxic than all of its metabolites, while on the contrary, according to Eq. (4), all of the 13 metabolites and degradation products of fenthion were expected to be more toxic than the parent compound. The acquired toxicity based on QSAR containing log
Based on the results of the current study, it appeared that fenthion can be highly toxic to the marine microalgal strain