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Comparative Toxicity of Three Organic Acids to Freshwater Organisms and Their Impact on Aquatic Ecosystems

March 15, 2006

By Saha, N C; Bhunia, F; Kaviraj, A

ABSTRACT

The comparative toxicity of lactic acid, acetic acid, and benzoic acid to tilapia (Oreochromis mossambicus), cladoceran Crustacea (Moina micrura), and oligochaete worm (Branchiura sowerbyi) were determined using static bioassay tests. Worms were found most sensitive to all the acids whereas the cladoceran was found most resistant to lactic acid and the fish most resistant to acetic acid and benzoic acid. The 96h LC^sub 50^ values of lactic acid, acetic acid, and benzoic acid, were, respectively, 257.73, 272.87, and 276.74 mg L^sup 1^ for O. mossambicus; 329.12, 163.72, and 71.65 mg L^sup -1^ for M. micrura and 50.82, 14.90, and 39.47 mg L^sup -1^ for B. sowerbyi. Tilapia lost appetite at sub-lethal concentrations as low as 2.18 mg L^sup -1^ lactic acid, 1.26 mg L^sup -1^ acetic acid, and 13.84 mg L^sup -1^ of benzoic acid. Growth and reproduction of the fish were affected following 90-day chronic exposure to sub-lethal concentrations of the acids. Minimum effective concentration of the acids that significantly reduced food conversion efficiency (FCE), percent increase of weight, specific growth rate, yield and fecundity of the fish were 2.18, 1.47, and 3.95 mg-L^sup -1^ of lactic acid, acetic acid, and benzoic acid, respectively. Effects of acetic acid and benzoic acid on FCE, weight increase, and yield were not significantly different from each other whereas lactic acid produced different effects from acetic acid as well as benzoic acid. Mean values of dissolved oxygen, primary productivity, and plankton populations of the test medium significantly reduced from control at 16.94 mg L^sup -1^ lactic acid, 16.79 mg L^sup -1^ acetic acid, and 13.84 mg L^sup -1^ benzoic acid.

Key Words: toxicity, organic acids, fish, crustaceans, worms, freshwater.

INTRODUCTION

Apart from daily household uses, the lactic acid, acetic acid, and benzoic acid are used by several industries and are discharged into the environment. Lactic acid is widely used in food processing industries (Budavari 1989) and is also commonly found in the effluent of many other industries such as cellulose (Walden et al. 1986) and pulp mill (Hruitford et al. 1975). Acetic acid is also identified in significant amounts in the industrial effluents like pharmaceuticals, cellulose, and pulp mill (Walden et al. 1986). Benzoic acid is commonly used for preserving foods, fats, fruit juices, alkaloid solutions, and so on, for curing tobacco, and as anti-fungal agent in pharmaceutical industries (Sittig 1985; Budavari 1989). It is also used as an herbicide (Caux et al. 1993) and is frequently found in the effluents of coal refining, paper and pulp mill, and in agricultural runoff (Kumaran 1993). Benzoic acid, although not properly quantified, is detected in surface water in many parts of Europe (URLl). Ground water near landfills receiving rural, municipal (domestic), and industrial wastes often show a benzoic acid up to 0.21 g L^sup -1^ (Guardiola et al. 1989). Stuermer et al. (1982) detected 16-860 g L^sup -1^ of benzoic acid in ground water at a coal gasification site. Leachates from foundry waste have been found to contain 200-400 g L^sup -1^ benzoic acid (Ham et al. 1989).

Little is known about the toxicity of these organic acids to aquatic organisms and their impact on physico-chemical parameters of water (Caux et al. 1993; Spencer and Ksander 1995). However, several reports indicate that these organic acids, if ingested or accumulated, produce several disorders to humans (Sittig 1985; Budavari 1989). This study was undertaken to evaluate relative toxicity of these organic acids to some freshwater organisms including a fish and two invertebrates and impact of these acids on physico-chemical parameters of water.

MATERIALS AND METHODS

Test Organisms

Test organisms used in the bioassay were a cichlid fish, tilapia ( Oreochromis mossambicus), a cladoceran Crustacea (Moina micrura) and an oligochaete worm (Branchiura sowerbyi). Only adult tilapia (mean length ranging from 102.50 to 77.53 mm, mean weight ranging from 11.83 to 7.73 g) was used for 96 h acute toxicity test, and feeding test, whereas juvenile tilapia (mean length 52.41 5.6 mm, mean weight 1.55 0.21 g) was used for 90 d chronic toxicity test. For all the tests both sexes of fish were used at random. When adult tilapia was used, gravid females were excluded from the test stock. The cladoceran (mean length 0.09 mm and mean breadth 0.06 mm) and the worm (mean length 20 7 mm and mean weight 2 1 mg) were used only for the 96 h acute toxicity test. Fish specimens were procured from local hatcheries whereas the cladoceran and worm were collected from local unpolluted sources. All the test organisms were acclimatized to the test condition for 96-192 h before they were used.

Test Chemicals

Analytical grade lactic acid (molecular formula: CH^sub 3^CHOHCOOH; molecular weight 100; colorless liquid; purity 88%), acetic acid (molecular formula: CH^sub 3^COOH; molecular weight 60.05; colorless liquid with a pungent vinegar like odor; purity 99%) and benzoic acid (molecular formula: C^sub 7^H^sub 6^O^sub 2^; molecular weight 122.12; creamy white crystal; purity 99%) were procured from E. Merck, India. Required quantity of an acid was taken out either by micropipette or by weighing and was treated directly into the test water.

Bioassays

Three types of static bioassays, two in the laboratory and one in an outdoor field, were conducted in this investigation. The laboratory bioassays include acute toxicity bioassay and feeding bioassays whereas the chronic toxicity bioassays were made in the outdoor field. Deep (300 feet underground) tube well water stored in an overhead tank was used as diluent medium (pH 7.21 0.11; free CO2 2.7 0.16 mg L^sup -1^; D.O. 5.3 0.70 mg L^sup -1^; alkalinity 172 6.78 mg L^sup -1^ as CaCO^sub 3^ and hardness 100 7.10 mg L^sup – 1^ as CaCO^sub 3^) for all the bioassays.

Acute toxicity bioassays

Acute toxicity bioassays for the cladoceran and the worm were conducted in 500 ml beakers whereas those for fish were carried out in 15 L glass aquaria. A beaker contained 300 ml water and 10 specimens either the cladoceran or the worm whereas an aquarium contained 10 liters of water and four fish. A set of four aquaria or beakers was exposed to a treatment thereby making four replicates for each of the several treatments used for acute toxicity bioassays (Table Ia). Mortality of the test organisms was recorded every 24 h and dead organisms were counted and removed from the experimental container immediately to avoid depletion of dissolved oxygen. The test medium was replaced every 24 h by fresh water and the desired quantity of respective acid was immediately added to the water to assure a constant concentration of the concerned acid in the solution and also to avoid other abiotic factors interfering with the animals’ performance. Similar techniques of replacement of test medium were adopted in the acute toxicity bioassays of aniline for tilapia (Bhunia et al. 2003). Total mortality of the animals recorded up to 96 h was used to calculate concentrations of the test chemicals at which 50% of the animals died (96 h LC^sub 50^) and its 95% confidence limits by probit analysis (Finney 1971). All the recommendations for static bioassay given by APHA (1995) were strictly followed.

Feeding bioassays

Feeding tests were also made in the laboratory for 96 h in 15 L glass aquaria each containing 10 L of water and 3 fish. A set of 20 aquaria, arranged in 5 different blocks each with 4 aquaria in a fully randomized block design (Gomez and Gomez 1984), was exposed to 5 treatments of each acid thereby making 4 replicates for each treatment (Table Ib). The test organisms were not fed 2 d before the start of the test to avoid interference by excretory substances on the toxicity of the solution (Verma et al. 1980). Live earthworms were cut into pieces and were given to the fish as food. The food was given daily at 8:00 AM and the fish were allowed to feed for 4 h. Unconsumed food pieces were collected from the aquarium and weighed. Weight of the food consumed was calculated from the total weight of food given minus the weight of food left unconsumed.

Table 1a. Concentrations of lactic acid, acetic and benzoic acid used in the acute toxicity bioassays.

Chronic toxicity bioassay

Chronic toxicity tests (90 d duration) were conducted in outdoor earthen vats each having a surface area of 0.65 m^sup 2^ and a capacity of 60 L water. Five kg uncontaminated soil was placed in the bottom of each vat. It was then filled with water and 325 g cow dung was applied to each vat. The vats were kept in this condition for one month in order to grow sufficient plankton, which served as natural food for fish. Such conditioned vats show minimum percolation of water and have been found suitable for evaluating chronic toxicity of chemicals (Saha and Kaviraj 1996). Water temperature of the vats ranged from 20-30C during the experiment. After conditioning, each vat was stocked with 15 acclimatized fingerlings of tilapia. Test chemicals were added 24 h after the stocking. Five treatments, including a control, were used for each acid (Table 1b).

In addition to the natural foo\d grown in the vat, the stocked fish were fed a mixture of rice bran and mustard oil cake (1:1) 6 d a week. Initially food ration was provided at the rate of 5% of the stocking weight. A 10% increase in the ration was made every 15 d.

Table 1b. Concentrations of lactic acid, acetic acid, and benzoic acid used in feeding and chronic toxicity bioassays.

pH, free CO2, total alkalinity, hardness, dissolved oxygen, primary productivity, phytoplankton, and zooplankton populations in the test medium were measured at every 15 d interval during the bioassay according to APHA (1995). Behavior and survival of fish were examined daily at 8:00 AM and 4:00 PM. Fish were sampled at the end of the experiment (90 d) and length, weight visceral weight, and gonad weight of the sampled fish were recorded. Final biomass was used to estimate the yield of fish in each treatment. Formulae used to calculate various parameters of growth and reproduction of fish were adopted from LeCren (1951), Brown (1975), and Bagenal (1978):

* Condition factor (K) = (W/L^sup 3^) 100, where W is the observed body weight of fish (g) and L is the body length of fish.

* Gastrosomatic index (GSI) = (V/W) 100, where V is the visceral weight of the fish (g) and W is the observed body weight of fish (g).

* Maturity (Gonadosomatic) index (MI) = (G/W) 100, where G is the gonad weight offish (g) and W is the observed body weight of fish (g).

* Fecundity = total number of ripening eggs/female.

* Percent increase in weight = (W^sub 2^-W^sub 1^)/W^sub 1^ 100, where W^sub 1^ is the initial weight of the fish (g) and W^sub 2^ is the final weight of the fish (g).

* Specific growth rate (SGR) %day^sup -1^ = {(log^sub e^ W^sub 2^ – log, W^sub 1^) / T} 100, where log^sub e^ W^sub 1^ is the natural logarithm of initial body weight of fish (g). log, W^sub 2^ is the natural logarithm of final body weight of fish (g) and T is the time interval.

* Food conversion efficiency (FCE) % = (weight gain/food given) 100, where weight gain = final weight offish (g) – initial weight offish (g).

Statistical Methods

Acute toxicity

Repeated measure ANOVA was performed on arcsin transformed mortality data followed by Duncan’s multiple range test (DMRT) to evaluate significant difference between the concentrations of the acids tested. The LC^sub 50^ values were also compared between the acids according to the procedures outlined by APHA (1995).

Feeding bioassay

Data from the feeding bioassays were subjected to single factor ANOVA, separately for each acid, followed by Duncan’s multiple range test to compare significant difference between the treatment means.

Chronic toxicity bioassay

Data for each parameter were subjected to single factor ANOVA followed by Duncan’s multiple range test to compare significant difference between treatment means. To compare the effects between the acids all data from the chronic toxicity bioassays were subjected to multivariate analysis on full factorial design using the type of acid and their treatments as fixed factors and the growth parameters or limnological parameters as dependent variables (Gomez and Gomez 1984).

Table 2. 96 hr LC^sub 50^ (mg . L^sup -1^) with 95% confidence limit of lactic acid, acetic acid, and benzoic acid to fish, plankton, and worm.

RESULTS

Acute Toxicity Bioassays

LC^sub 50^ values of lactic acid, acetic acid, and benzoic acid for the fish ( Oreochromis mossambicus), cladoceran (Moina micrura), and worms (Branchiura sowerbyi), determined from the acute toxicity tests, have been summarized in Table 2. The LC^sub 50^ values indicate that the worms are the most sensitive to all the organic acids whereas the cladoceran is most resistant to lactic acid and the fish most resistant to acetic acid and benzoic acid. The results of the repeated measures ANOVA and the Duncan’s multiple range test show that the minimum doses of the organic acids that caused a significant mortality of the fish, cladoceran, and worms were, respectively, 60.50, 290.40, and 38.72 mg L^sup -1^ for lactic acid, 251.88,125.94, and 12.59 mg L^sup -1^ for acetic acid, and 240.00, 69.60, and 37.20 mg L^sup -1^ for benzoic acid. LC^sub 50^ values of the acids for both the cladoceran and the worms varied significantly between the acids whereas the LC^sub 50^ values of the acids for the fish did not vary significantly between the acids.

Whitish lesions appeared on the skin and fins offish after few hours of exposure to the high concentrations of lactic acid (242 mg L^sup 1^ or more ) and acetic acid (262.4 mg L^sup -1^ or more). Fish exposed to high concentrations of all the acids showed symptoms of respiratory distress such as frequent surfacing, abnormal opercular movement, grasping, release of air bubbles from mouth, and loss of equilibrium. Blood vomiting was also recorded before death offish exposed to high concentration of benzoic acid.

Worms exposed to higher concentration of lactic acid and acetic acid reacted sharply by coiling their body. Gradually the body was fragmented resulting in death of the worms. Worms exposed to higher concentrations of benzoic acid initially showed brisk movement followed by sluggishness and death. Cladocerans exposed to all acids showed decreasing swimming activity before death.

Feeding Bioassays

Changes in the feeding rate of tilapia exposed to different organic acids have been presented in Table 3. Single factor ANOVA (F^sub (4,15)^ = 18.82, p

Table 3. Food consumed (g/fish) ) by tilapia exposed to the organic acids. Values are mean of four replicates SD. Different superscript letters indicate significant difference between the treatments (DMRT; p

Chronic Toxicity Tests

Behavior, growth, and reproduction

There was no mortality offish during chronic toxicity bioassay of the acids. There was also no apparent change in behavior and color of the exposed fish. Control fish showed 214% increase in weight after 90 days period of growth. Food conversion efficiency (FCE), specific growth rate (SGR), yield and fecundity of the control fish were respectively 43.76 %, 1.27 % day^sup -1^, 254 g.M^sup 2^, and 218. These values and maturity index (both male and female; 1.22 and 8.22, respectively, in control) significantly reduced and gastro- somatic index (GSI; 7.87 in control) significantly increased under sub-lethal concentrations of the acids. The minimum effective concentration of lactic acid, acetic acid, and benzoic acid that caused a significant reduction in FCE, SGR, percent increase in weight, yield, and fecundity over control, were, respectively, 2.18 mg L^sup -1^ (T2), 1.47 mg L^sup -1^ (T3), and 3.95 mg L^sup -1^ (T2). Condition factor (K) did not show any variation between treatments. Maturity index of male significantly reduced in all concentrations of the acids tested (T2 to T5), whereas that of female decreased only in concentrations 5.08 to 25.41 mg L^sup -1^ oflacticacid (T3 to T5), 16.79 to 27.29 mg L^sup -1^ of acetic acid (T4 to T5), and 7.16 to 27.67 mg/L of benzoic acid (T3toT5).

Effects of acetic acid and benzoic acid on FCE, weight increase, and yield were not significantly different from each other whereas lactic acid produced effects different from acetic acid as well as from benzoic acid (Table 4). Effects of acetic acid on fecundity of the fish were different from both lactic acid and benzoic acid, whereas lactic acid and benzoic acid produced similar effects on fecundity. There was no significant difference in effects between the acids on condition factor (K), and maturity index of both male and female. However, all the parameters of fish showed significant difference between the treatments. Further details of the chronic toxicity data are available from the authors.

Limnological parameters

Free CO2 of water increased, whereas total alkalinity, dissolved oxygen (DO), primary productivity, phytoplankton and zooplankton abundances of water reduced significantly at higher concentrations of acetic acid (16.79 and 27.29 mg L^sup -1^) and benzoic acid ( 13.84 and 27.67 mg L^sup -1^). Similar changes were also recorded at higher concentrations of lactic acid (16.94 and 25.41 mg L^sup -1^) except zooplankton population, which did not show any significant variation (p > .05) over control under lactic acid treatment. pH also did not vary significantly (p > .05) from control in lactic acid and benzoic acid treated water. Higher concentration of acetic acid (16.79 and 27.29 mg L^sup -1^), however, significantly reduced pH of water over control. No significant changes in hardness of water were observed under any treatment of the acids. Comparing the effects between the acids it was observed that only data of free CO2 and the phytoplankton population differed significantly between the acids.

Table 4. Results of multivariate analysis on observations of growth and limnological parameters (dependent variables) using types of acid (LA, AA, BA) and treatments as the fixed factors.

DISCUSSION

Although sensitivity of aquatic animals to toxicants varies widely among species, the invertebrates are generally found more sensitive to toxicants than vertebrates (Kaviraj and Das 1990; Das and Kaviraj 1999). In our earlier studies on the toxicity of aniline and methanol we also observed that the crustacean Moina micrura was most sensitive to th\ese toxicants, but the oligochaet worm Branchiura sowerbyi in spite of being an invertebrate was least sensitive to these toxicants (Bhunia et al. 2003: Kaviraj et al. 2004). Contrary to these findings, results of the present investigation indicate that oligochaet worms are more sensitive to acetic acid, lactic acid, and benzoic acid than fish. Saha and Kaviraj (1996) also observed oligochaet worms more sensitive to tannic acid than fish. Mechanism of toxicity of lactic acid, acetic acid, and benzoic acid is not known. Lethal doses of the acids used in the acute toxicity bioassays resulted in reduction of pH of water (28 to 37% for lactic acid, 10 to 40% for acetic acid, and 12 to 60% for benzoic acid; data not presented) as compared to control water, within 24 hours of treatment. However, reduction in pH of water alone could not account for the toxicity of these acids. All the organic acids were highly toxic to the worm and became lethal at very small concentration in which pH of water changed little, whereas a high concentration of lactic acid, which rendered maximum reduction in pH of water, produced least toxicity to Moina micrura (see Table 2).

Comparing sensitivity of fish to different organic acids it is observed that LC^sub 50^ values of the organic acids for tilapia as recorded in the present investigation (257.73 to 276.74 mg L^sup – 1^) are close to the LC^sub 50^ values of benzoic acid reported for freshwater fish by Caux et al. ( 1993) ; but higher than the LC^sub 50^ values (ranging from 40 to 12800 g L^sup -1^) of many other organic acids reported for fish (Takagi et al. 1982; Willard 1983; Viswaranjan et al. 1988). LC^sub 50^ value of benzoic acid to crustacean M. micrura observed in the present investigation are also comparable to the LC^sub 50^ values of benzoic acid to other invertebrates (Verscheuren 1977).

Lethal doses of the organic acids produced irritating effects on fish. It was evident from frequent surfacing, grasping, and brisk opercular movements of the fish. Whitish lesions observed in the skin and fins offish exposed to high concentrations of lactic acid and acetic acid are probably the result of burning effects, the most common effects of acids recorded for human skin and eyes (Sittig 1985). Literatures are also available to support irritation caused to fish by acute exposure of acetic acid (Sittig 1985; Budavari 1989). Respiratory distress was also responsible for the unusual behavior offish. Blood vomiting before death of the fish following acute exposure to benzoic acid indicated acute diarrhea and rupturing of finer blood vessels in the gastrointestinal tract offish. Similar effects are found in humans after ingestion of benzoic acid (Budavari 1989).

Loss of appetite, reduction in growth, and discrepancy in reproduction parameters indicate that lactic acid and benzoic acid are capable of producing chronic toxic effect on fish even in small concentration. Although very few literatures are available on chronic toxicity of organic acids to fish, Saha and Kaviraj (1996) observed that sub-lethal concentration of tannic acid, an organic acid, also produced adverse effects to tilapia following chronic exposure . Extreme reduction in yield of fish (51.24%) was recorded at 25.41 mg L^sup -1^ lactic acid concentration. Reduction in growth and reproduction parameters following a 90 d exposure to sub-lethal concentrations of lactic acid (2.18 to 25.41 mg L^sup -1^), acetic acid (1.47 to 27.29 mg L^sup -1^) and benzoic acid (3.95 to 27.67 mg L^sup 1^) has important ecological implications. Permissible limits of the organic acids in water are not known. The present study recorded harmful effects on fish only at an initial treatment of 2.18 mg L^sup -1^ lactic acid, 1.47 mg L^sup -1^ acetic acid, and 3.95 mg L^sup -1^ benzoic acid. Although lactic acid and acetic acid are frequently detected in the effluents of several industries, quantitative data about their levels in the environment are poorly documented. But there are reports that benzoic acid can concentrate in water to a very high concentration (Stuermer et al. 1982; Ham et al. 1989). In the light of the present study a persistent level of 860 g L^sup -1^ of benzoic acid in water, as observed by Stuermer et al. (1982) in ground water near a coal gasification site, would definitely render harmful effects on growth and reproduction of fish.

Results of chronic toxicity tests also indicate that the sublethal concentrations of lactic acid (16.94 mg L^sup -1^), acetic acid (16.79 mg L^sup -1^), and benzoic acid (13.84 mg L^sup -1^) may reduce DO, primary productivity, and phytoplankton population. Abrupt reduction in DO often produces a negative impact on the general productivity of water body (Sudhan et al. 1984) because it leads to reduced growth rate and sub-optimal food conversion efficiency offish (Stickney 1994). Caux et al. (1993) reported growth inhibition of freshwater algae exposed to benzoic acid as herbicide dicomba (100 to > 10,000 g l^sup -1^). Growth of aquatic weed Hydrilla verticillata had also been found to be inhibited by acetic acid (Spencer and Ksander 1995). Therefore, changes in the limnological parameters (particularly DO), primary productivity, and abundances of phytoplankton populations caused by chronic exposure of the acids, as observed in the present investigation, might have produced a negative effect on the growth of the fish.

ACKNOWLEDGMENTS

The authors are thankful to the Head, Department of Zoology, University of Kalyani and Principal, Jhargram Raj College, for providing necessary facilities for this research.

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N. C. Saha,1 F. B\hunia,2 and A. Kaviraj3

1 Post Graduate Department of Zoology, Presidency College, Kolkata 700073, India; 2 Jhargram Raj College, Jhargram, Midnapore, W.B., India; ‘Department of Zoology, University of Kalyani, Kalyani 741235, W.B., India

Received 5 February 2005; accepted 20 May 2005.

Address correspondence to Dr. A. Kaviraj, Department of Zoology, University of Kalyani, Kalyani, 7412235, W.B., India. E-mail: anilava@vsnl.net

Copyright Taylor & Francis Ltd. Feb 2006




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