Abstract
Static renewal bioassays were conducted in the laboratory and in outdoor artificial enclosures to evaluate toxic effects of methanol to one teleost fish and two aquatic invertebrates and to limnological variables of aquatic ecosystem. Ninety-six-hour acute toxicity tests revealed cladoceran crustacea Moina micrura as the most sensitive to methanol (LC50, 4.82 g/L), followed by freshwater teleost Oreochromis mossambicus (LC50, 15.32 g/L) and oligochaete worm Branchiura sowerbyi (LC50, 54.89 g/L). The fish, when exposed to lethal concentrations of methanol, showed difficulties in respiration and swimming. The oligochaete body wrinkled and fragmented under lethal exposure of methanol. Effects of five sublethal concentrations of methanol (0, 23.75, 47.49, 736.10, and 1527.60 mg/L) on the feeding rate of the fish and on its growth and reproduction were evaluated by separate bioassays. Ninety-six-hour bioassays in the laboratory showed significant reduction in the appetite of fish when exposed to 736.10 mg/L or higher concentrations of methanol. Chronic toxicity bioassays (90 days) in outdoor enclosures showed a reduction in growth, maturity index and fecundity of fish at 47.49 mg/L or higher concentrations of methanol. Primary productivity, phytoplankton population, and alkalinity of water were also reduced at these concentrations. Chronic exposure to 1527.60 mg/L methanol resulted in damages of the epithelium of primary and secondary gill lamellae of the fish. The results revealed 23.75 mg/L as the no-observed-effect concentration (NOEC) of methanol to freshwater aquatic ecosystem.
Methanol is an organic aliphatic hydroxy solvent widely used as a raw material in a number of industries such as manufacture of formaldehyde, pesticides, photographic film, plastic, textile, soap, artificial leather, etc., and as solvent for ink, resins, adhesives, and dyes (Sittig 1980; Walden, McLean, and McCauley 1986). It is also used for extraction of quinidine in quinine factories (Saha, Kaviraj, and Som 1988) and as an ingredient in paints and varnish removers, cleaning and dewaxing preparations, spirits, etc. (Budavari 1989). As a result, it is found in the effluent of many industries.
Methanol poisoning in man and some mammals has been studied in detail (Anderson, Shuaib, and Becker 1987; Andrews et al. 1987). These studies indicate that methanol is metabolized primarily in the liver to formaldehyde, which is further oxidized to formic acid or formate depending upon pH. Many species can detoxify formate to carbon dioxide. Acute and short-term toxicities of methanol vary greatly between different species, toxicity being highest in species with relatively poor ability to metabolize formate (Johlin et al. 1987). Metabolism of methanol in fish is poorly known. A search of literatures indicates that methanol is lowly toxic to aquatic organisms. Ninety six hours LC50 value of methanol to fish varies between 15,400 to 29,400 mg/L (Veith, Call, and Brooke 1983; Poirier et al. 1986), whereas that of blue mussel Mytilus edulis is 15,900 mg/L (Helmstetter, Gamerdinger, and Pruell 1996). Ten-day LC50 value of green algae Chlorella pyrenoidosa was found as 28,501 mg/L (Stratton and Smith 1988). However, a scant literature is available on long-term effects of sublethal concentrations of methanol to fish and aquatic ecosystem. Methanol is quickly degraded in the environment by photo oxidation and biodegradation process. But an ecosystem, perturbed by lethal or sublethal concentration of a toxicant, takes time to restore to its normal functioning even after the toxicant is degraded or removed from the system. Sublethal effects of methanol, particularly on fish and aquatic ecosystem, are poorly documented. In the present investigation, attempts were made to determine 96-h lethal concentrations of methanol for freshwater teleost fish Oreochromis mossambicus, cladoceran crustacea Moina micrura, and oligochaete worm Branchiura sowerbyi and also to determine long term effects of methanol at sublethal concentrations to the fish and some ecosystem variables. The study was not designed to mimic environmental level of methanol but to evaluate sublethal effects of methanol that may continue in an ecosystem for long period.
MATERIALS AND METHODS
Test Organisms
Test organisms used in the bioassays were an omnivorous fish tilapia (Oreochromis mossambicus; Order: Perciformes; Family: Cichlidae), a planktonic crustacea (Moina micrura; Order: Cladocera; Family: Daphnidae), and a bottom dwelling worm (Branchiura sowerbyi; Class: Oligochaeta; Family: Tubificidae), representing three different trophic levels of aquatic ecosystem. These organisms are easily available in India and are widely used as test organisms in laboratory and outdoor bioassays of toxicants (Kaviraj and Konar 1982; Saha, Kaviraj, and Som 1988; Bhunia, Saha, and Kaviraj 2000). Only adult tilapia of both sex were used for 96-h acute toxicity tests, respiratory tests, and feeding tests, whereas fingerlings were used for chronic toxicity tests. The crustacean and the oligochaete worm were used only for 96-h acute toxicity tests. Test fish tilapia was procured from local hatcheries, whereas plankton and worm were collected from local unpolluted sources. All these test organisms were acclimatized to the test condition for 96 h to 192 h before their use.
Test Chemicals
Analytical grade methanol (CH3OH, Ranbaxy, India; molecular weight 32.04; weight per milliliter at 20°C is 0.790 to 0.793 g) was used as the test chemical. It was available in liquid form and was treated directly to the test medium. Required volume of the chemical added to the test medium was converted into weight and the treatment was expressed in terms of weight per volume.
Bioassays
Static renewal bioassays were used for both 96-h tests in the laboratory (acute toxicity test and feeding test) and 90-days chronic toxicity tests in outdoors. Deep tube well water stored in an overhead tank (pH 7.34 ±0.12, free CO2 2.61 ±0.19 mg/L, DO 6.45 ±0.56 mg/L, alkalinity 182 ±7.94 mg/L as CaCO3, hardness 109 ±7.2 mg/L as CaCO3) was used as the diluent for all bioassays. Procedures outlined in American Public Health Association (APHA) (1989) for static renewal bioassays were followed.
Acute Toxicity Test
Acute toxicity tests for the crustaceans and the worms were run in 500-ml beakers with 300 ml water in each beaker. Ten crustaceans (mean length 0.09 ±0.01 mm) or worms (mean length 20 ±7 mm; mean weight 2.05 ±1.25 mg) were stocked in each beaker and four replicates of beaker were used for each concentration of methanol. Acute toxicity tests for fish were conducted in 15-L glass aquaria with 10 L of water. Four fish (mean length 78.5 ±5.4 mm; mean weight 7.8 ±0.8 g) were stocked in each aquarium and four such aquaria were exposed to each concentration of methanol including a control. All the test organisms were acclimatized to the test condition for 48 h before any treatment of methanol was made. No food was provided during the bioassay to avoid interference of excretory product of fish with the test chemical (Verma et al. 1980). Mortality was recorded every day in the morning, dead organisms were removed, and water of the experimental aquaria was siphoned out and replaced by fresh water with desired quantity of methanol added to it. Before the water was siphoned out, pH, free CO2, and dissolved oxygen were measured in samples of water. Cumulative mortality of the test organisms after 96 h was used to estimate LC50 values by probit analysis (Finney 1971).
Changes in the respiratory rate of fish exposed to different concentrations of methanol were evaluated from the opercular movements. Opening and closing of operculum by fish was taken as one unit of opercular movement. Total number of opercular movements per minute was recorded for fish. Ten such observations were made at random for each concentration every 24 h. Ninety-six-hour mean opercular movements per minute were recorded for each concentration of methanol tested.
Feeding Test
Ninety-six-hour static renewal bioassays were made in the laboratory to evaluate feeding rate of fish exposed to methanol. The bioassays were made in 15-L glass aquaria each containing 10 L of water. Three adult fish (mean length 82.5 ±5.7 mm, mean weight 8.7 ±1.5 g) were stocked in each aquarium. Five sublethal concentrations of methanol including a control (0.00, 23.75, 47.49, 736.10, and 1527.60 mg/L) were tested. The sub-lethal concentrations were derived from the 96-h LC50 values of methanol to the crustacea and the fish. The two lower sub-lethal concentrations were respectively 0.5% and 1.0% of the LC50 values for the crustacea and the two higher sublethal concentrations were respectively 5% and 10% of the LC50 values for the fish. The final concentrations were adjusted to the actual volume of methanol added to the medium, thereby making the actual sublethal concentrations as 0.49% and 0.98% of the LC50 values for the crustacea and 4.81% and 9.97% of the LC50 values for the fish. Four aquaria were exposed to each sublethal concentration in randomized block design so that there were four replicates for each concentration of methanol tested. Live earthworms were cut into uniform pieces and were given fresh to fish as food. The food was given daily at 8 AM and the fish were allowed to feed for 4 h. Unconsumed food pieces were removed, to avoid any decomposition hazard in the test medium, and weighed. Water was siphoned out every 24 h and replaced by fresh water with desired quantity of methanol added to it. Weight of the food consumed was calculated from the total wet weight of food given minus the weight of food left unconsumed. Total amount of food consumed by fish in control was taken as 100%. Percentage of food consumed by fish in each sublethal concentration of methanol was determined relative to control.
Chronic Toxicity Test
Chronic toxicity bioassays were conducted in 60-L earthen vats for 90 days under natural environmental conditions. Temperature of water ranged from 20°C to 30°C during the experiment. Vats were arranged in five blocks, each with four vats as per randomized complete block design (Gomez and Gomez 1984), thereby giving four replicates for each concentration of methanol tested. All five sublethal concentrations of methanol used in the feeding test (0.00, 23.75, 47.49, 736.10, and 1527.60 mg/l) were also used in the chronic toxicity bioassays. The vats were prepared thoroughly before the start of experiment. The bottom of each vat was provided with 3 cm thick soil and was filled with water. They were kept in this condition for about 1 month in order to allow growth of plankton population. A fixed level of water was maintained in the vat through regular supply of water and monitoring. Such conditioning of earthen vats allows minimum percolation of water (Saha and Kaviraj 1996; Bhunia, Saha, and Kaviraj 2000). When sufficient plankton population grew in the vats to serve as natural food for fish, each tank was stocked with 15 fingerlings of tilapia (mean length 51.7 ±4.3 mm; mean weight 1.4 ±0.4 g). Two days after the stocking, the vats were treated with five sublethal concentrations of methanol as mentioned above. Every week, 10% of test medium was replaced by fresh water containing desired quantity of methanol. In addition to the natural food, the stocked fish were fed a mixture of rice bran and mustard oil cake (1:1) 6 days a week at 5% of the stocking weight. A 10% increase of ration was provided every fortnight.
pH, free CO2, total alkalinity, hardness, dissolved oxygen, gross primary productivity, and plankton population in the test medium were measured at every 15 days during the bioassays following procedures of APHA (1989). Mortality and abnormality in the behavior of the fish, if any, were examined twice (at 8 Condition factor (K ):
(b) Relative condition factor (Kn):
Gastrosomatic index (GSI):
Maturity (gonadosomatic) index (MI):
Fecundity = total number of ripening eggs/female. Percent increase in length =[(L2 − L1)/L1]× 100where L
1 = initial length of fish (mm); L
2 = final length of fish (mm) Percent Increase in weight =[(W
2−W
1)/W
1]× 100where W
1 = initial weight of fish (g); W
2 = final weight of fish (g) Specific growth rate (SGR) %/day:
Food conversion ratio (FCR):
Food conversion efficiency (FCE)%:
Statistical Analyses
All data except those of acute toxicity test were analyzed for significance of variation. Data of opercular movements were analyzed by two-way analysis of variance (ANOVA), taking days of exposure and concentrations of methanol as independent variables. Data of growth, reproduction, and limnological parameters (90-days mean values) were subjected to single-factor ANOVA. If the data significantly varied (p < .05) Duncan’s Multiple Range Test (DMRT) was employed to test significance of difference among concentrations of methanol tested. Evaluations were made at 5% level of significance (Gomez and Gomez 1984).
RESULTS
Acute Toxicity of Methanol
Ninty-six-hour LC50 values and their 95% confidence limit of methanol to the fish, crustacean, and worm are given in Table 1. These values indicate that the tolerance of the animals to the methanol lies in order worm > fish > crustacean. Fish exposed to lethal concentrations of methanol exhibited hyperactivity and convulsion. They also showed signs of suffocation. In contrast to normal behavior of control fish, treated fish frequently visited the surface. Worms wrinkled at high concentration of methanol and immediately died. In lower concentrations of methanol, death of the worms was delayed. The tail portion affected first followed by mucous secretion and death. Only minor variations from initial values were observed in pH, free CO2, and dissolved oxygen of water in control and methanol treated aquaria.
As shown in Figure 1A , 96-h mean opercular movements of fish decreased with the increase of concentrations of methanol. Two-way ANOVA on data of opercular movements, taking days of observation and concentrations of methanol as independent variables, showed that opercular movements of fish exposed to methanol significantly varied (p < .01) among both days and concentrations of methanol. The opercular movements and concentrations of methanol showed a negative straight-line relationship at 48 h (y = 148.33 − 0.002x; r = − .65; p = .06), 72 h (y = 145.25 − 0.002x; r = − .74; p = .02), and 96 h (y = 147.02 − 0.002x; r = − .79; p = .03). The correlation between opercular movements and concentrations of methanol at 24 h was insignificant (r = − .25; p = .32) (Figure 1B ).
Effects on Feeding Rate of O. mossambicus
Figure 2 shows that feeding rate of tilapia reduced with the increase of concentration of methanol. Single-factor ANOVA followed by DMRT showed that up to 47.49 mg/L methanol concentration, there was no significant alteration in the feeding rate of the fish as compared to control (p > .05). Feeding rate significantly reduced at methanol concentrations 736.10 and 1527.60 mg/L (p < .05). The reduction was severe (32.26% of control) at 1527.60 mg/L.
Chronic Toxicity of Methanol
There was no mortality of fish during outdoor bioassays. But the total yield of fish reduced significantly (p < .05) in three higher concentrations of methanol (47.49, 736.10, and 1527.60 mg/L). A severe reduction of yield (76.86%) was found in 1527.60 mg/L methanol treatment as compared to control (Tables 2 and 3). Mean length, mean weight, specific growth rate (SGR), and food conversion efficiency of fish also changed significantly in the higher concentrations of methanol. Gastrosomatic index (GSI), maturity index (MI) of both male, and female, and fecundity of fish also changed significantly in these concentrations. No significant change was observed for condition factor and relative condition factor of fish as compared to control.
The fish exposed to highest sublethal concentration of methanol (1527.60 mg/L) exhibited some histopathological changes in their gills. Cysts formed on the primary gill lamellae and intralamellar space of the primary gill epithelium increased. Secondary gill lamellae reduced in size or swelled due to edematous separation of the epithelium and hypertrophy of the epithelial cells. Water space in between two secondary lamellae decreased considerably, resulting in reduction of space for gaseous exchange in the gill. However, the pillar cell system remained mostly unaffected. The fish exposed to control and lower concentrations of methanol (23.75, 47.49, 736.10 mg/L) exhibited normal primary and secondary lamellae of the gills. The secondary lamellae were uniform in size, with stratified epithelial cells, blood capillary, and vacuoles. Water space in between two secondary lamellae was normal.
Limnological Parameters
Limnological parameters, except pH and hardness of water, showed significant variation among various concentrations of methanol. Mean values (90 days) of the limnological parameters have been recorded in Table 4. Free CO2 increased whereas dissolved oxygen, gross primary productivity, alkalinity, phytoplankton population, and zooplankton population reduced in high concentrations of methanol (736.10 to 1527.60 mg/L). No effect was observed at 23.75 mg/L methanol. At 47.49 mg/L concentration, alkalinity, gross primary productivity, and phytoplankton population reduced significantly as compared to control, whereas other parameters remained comparable to control.
DISCUSSION
LC50 values of methanol to aquatic organisms recorded in the present investigation indicate that Branchiura sowerbyi is more tolerant to methanol than Oreochromis mossambicus, whereas Moina micrura is least tolerant to methanol. LC50 value of methanol for bluegill Lepomis macrochirus (15400 mg/L) recorded by Poirier et al. (1986) is close to the value observed for tilapia in the present investigation. However, rainbow trout (LC50 20,100 mg/L) and fathead minnow (LC50 28,100 to 29,400 mg/L) have been found more tolerant than either bluegill or tilapia (Veith, Call, and Brooke 1983; Poirier et al. 1986). Methanol is an aliphatic hydroxy compound and is relatively less toxic to fish as compared to aromatic hydroxy compound phenol. LC50 of phenol to three Indian freshwater fish ranged from 12.53 to 39.40 mg/L (Verma et al. 1980).
There is a scant literature on the effects of methanol on invertebrate. Methanol caused sluggish movements with extended siphon and slow reflexes in mussel Mytilus edulis (Helmstetter, Gamerdinger, and Pruell 1996). Reactions of the worms to methanol as observed in the present investigation are similar to reactions observed in other aquatic invertebrates exposed to methanol (Tephly 1991; Helmstetter, Gamerdinger, and Pruell 1996). The worm Branchiura sowerbyi, like most of the freshwater oligochaetes, is a primary consumer of the detritus-based food chain of freshwater bodies. It feeds on dead organic matter, particles of detritus, algae, and other microorganisms trapped in the mucus secreted by the worms (Ward and Whipple 1963; Ruppert and Barnes 1994). Strands of mucus are visible in a dense population of the worm. The mucus on the body surface as well as the microorganisms associated with mucus are responsible for high tolerance of the worm to methanol. Mucus reduces permeability of the body surface to pollutants whereas methanol is widely dispersed and metabolized or degraded through microbial activity (Pawel et al. 2000).
Many researchers reported behavioral changes of fish due to methanol toxicity (Verma et al. 1980; Hlohowskyi and Chagnon 1991). Hyperactivity and convulsion of fish exposed to methanol indicate effects of methanol on central nervous system (CNS) of fish (Tephly 1991). Methanol also affects the gill tissue, resulting in respiratory distress of fish. This is evident from the frequency of opercular movements and excessive secretion of mucus from gill during acute exposure and reduction in the respiratory surface due to damage of gill epithelia after chronic exposure to methanol. Bucher and Hofer (1993) also indicated respiratory distress in methanol-exposed fish. Respiratory distress in methanol-treated fish may also result from increased level of CO2 in blood. Methanol is metabolized primarily in the liver by sequential oxidative steps to formaldehyde–formic acid or formate–CO2 (Anderson, Shuaib, and Becker 1987; Andrews et al., 1987). Formate is implicated as the main toxic compound of acute methanol poisoning. If animals fail to metabolize formate metabolic acidosis results. If formate is readily metabolized, CNS is depressed, resulting in comma, respiratory distress, etc. (Johlin et al. 1987; Tephly 1991). The primates develop increased blood formate, concentration following methanol exposure, but rodents do not develop such high concentration of formate because they have the enzyme system to metabolize formate which the primates are lacking (Smith and Taylor 1982). Metabolism of methanol in the body of fish is poorly known.
It is revealed from the present study that chronic exposure of even very low concentration of methanol (47.49 mg/L) may result in reduction of growth and impairment of reproductive performance of tilapia. Reduction in growth of fish exposed to methanol was revealed from the low percent of increase in length and weight, SGR, and yield. A reduction in weight of the fish also resulted in an increase in GSI value of the fish. Growth is directly related to feeding efficiency of fish. Feeding rate of tilapia is significantly reduced after 96-h exposure to a concentration of 736 mg/L methanol. Reduction of feeding efficiency even at much lower concentration of methanol after chronic exposure and subsequent effect on growth of fish is therefore not unusual. Reduced feeding rate of fish is a result of metabolic disorders. Although report of such metabolic disorders due to methanol toxicity is lacking in fish, acute metabolic acidosis, hyperamylasemia, and hyperglycemia as a result of methanol toxicity are reported in man (Pappas and Silverman 1982; Eckfeldt 1986). Pilat and Prokop (1975) reported that the specific growth rate of the fish Candida boidinii remained constant upto concentration of methanol (3% v/v). Beyond this level, the growth of fish was reduced significantly. Results of the present investigation show that the lowest concentration of methanol (47.49 mg/L equivalent to 0.06 ml/L) capable of reducing growth of tilapia is much lower than that observed by Pilat and Prokop (1975). This concentration of methanol also results in reduction in fecundity and maturity index of fish. Therefore, tilapia (Oreochromis mossambicus) is more sensitive to methanol than the fish Candida boidinii.
Significant reduction of dissolved oxygen, primary productivity, phytoplankton, and zooplankton populations are found at higher concentrations of methanol (736.10 to 1527.60 mg/L). The magnitude of alteration in limnological parameters is not alarming for tilapia but may be harmful to other sensitive species of fish and invertebrates.
CONCLUSION
The present study was not designed to mimic environmental level of methanol. Instead, efforts were made to evaluate impact of methanol to aquatic ecosystem in general. Apart from evaluating toxicity of methanol to three different aquatic organisms, representing three different trophic levels, efforts were made to evaluate impact of methanol on primary productivity, plankton population, and physicochemical parameters of aquatic ecosystem.
Results of acute toxicity study indicate that methanol is of low toxicity to aquatic organisms, particularly to higher trophic level organisms such as fish and scavenger such as oligochaet worm. Relatively high sensitivity of crustacean plankton to methanol indicates that it may affect the food chain and community function in the aquatic ecosystem even at low concentration. This is also confirmed from the chronic toxicity bioassays that show that methanol is capable of retarding the growth and reproduction of fish and reducing primary productivity, phytoplankton population, and alkalinity of water at a concentration of 47.49 mg/L (equivalent to 0.06 ml/L)—the lowest-observed-effect concentration (LOEC) of methanol. The concentration of methanol that produces no observed effect (NOEC) to freshwater ecosystem in chronic exposure is 23.75 mg/L (equivalent to 0.03 ml/L). Results of the present study is important to evaluate long-term effects of hydroxy compounds to aquatic ecosystem.
Footnotes
Figures and Tables
Acknowledgements
The authors are grateful to Principal, Jhargram Raj College, Jhargram, and Head, Department of Zoology, University of Kalyani, for providing necessary facilities for this research.