Introduction

Effluents are very important source of chemicals entering into aquatic ecosystem, deteriorating the water quality and thereby affecting the flora and fauna. There have been increasing trends towards use of biomonitoring to assess the water quality of resources/receiving water bodies [ ₁₂ ]. The analysis of a recognized set of water chemistry parameters is the most commonly used method of assessing water quality by pollution control boards/regulatory authorities. However, these parameters provide information about the chemical complexity but not their toxicity. Moreover, study of physico-chemical parameters of effluent does not throw any light on the synergistic or antagonistic effects on biological systems. Thus an adequate monitoring system should assess both the effects and distribution of pollutants [ ₃ ]. The chemical analysis identifies only the main chemical pollutants available while a bioassay (toxicity test) addresses pollutant bioavailability and response to total effect of actual potential [ ₄ ].

The traditional acute multi-concentration toxicity tests when applied for the determination of effluent is often recognized as Whole Effluent Toxicity (WET) test. The WET test is widely used in the USA, Canada and European countries, and is an important component of the environmental protection agencies of these countries for detecting and addressing the toxicity of industrial effluents/wastewaters. Determination of acute toxicity of the chemical products is one of the cornerstones of the general hazard assessment of the programs laid down by various environmental protection agencies. However, most of the regulatory agencies and research organizations are using the well-established conventional acute toxicity test with fish and daphnids in which survival/death/immobility is the most frequently monitored end point [ ₅₆₇ ].

Daphnids are routinely used as standard test organism in aquatic toxicity bioassay (acute and chronic) because of their rapid reproduction, sensitivity to chemical environment, and play key ecological role in the aquatic food chain by serving as an intermediate between primary producers and fish [ ₈ ]. Daphnia magna has been evaluated as a good organism to test effluent toxicity in textile wastewater [ ₉₁₀ ], dye and dyes wastewater [ ₁₁₁₂₁₃ ].

India is among one of the major producers of dyes and dyes intermediate from Asian region and meets the requirement of the world at large. There are about 900 dyes and dyes intermediate producing industries in India. Most of them are in small-scale unorganized industrial sector, and about five thousand dyes and dyes intermediate are in commercial production. The wastewater generated from dye and dye intermediate industries mainly have intense color having various shades of red, blue green, brown and black through the production of different color containing dyes and usually have high level of COD, BOD, acidity, chlorides, sulphates, phenoloic compounds and various heavy metals viz. copper, cadmium, chromium, lead, manganese, mercury, nickel, zinc etc. [ ₁₄ ]. Although it seems logical that dyes should be safer to the environment than neurotoxic insecticides [ ₈ ], little work has been done to determine the potential impact of dyes on the ecosystem or selected indicator sps. in the laboratory [ ₁₅₁₁₁₆ ]. Dyes, as they are intensively colored, cause special problems in effluent discharge and even small amount is noticeable. The effect is aesthetically more displeasing rather than hazardous, and can prevent sunlight penetration decreasing photosynthetic activity in aquatic environment. Although, some azo dyes that causes the effluent color have been implicated as being mutagenic/carcinogenic as well as toxic to aquatic life [ ₁₇₁₈ ].

Therefore in order to assess the effluent toxicity status of polluting industries traditional acute toxicity test (WET) was applied for dyes and dyes intermediate producing industries using Daphnia magna as a test model. The overall objective of this study was to assess the acute toxicity of industrial effluents of which seven are dyes and dyes intermediate producing industries and one is common effluent treatment plant which receives dyes effluents from more than fifty dyes producing small scale industries. Further, our aim was also to contribute towards the development of a more effective policy of pollution control and effluent regulation using bio-criteria derived from acute WET test results.

Materials and Methods

Grab samples of the effluents were collected at the point of outlet discharge from the wastewater treatment plants (WTPs) of seven different dyes and dyes intermediate producing factories (labeled as A, B, C, D, E, G and H) and a common effluent treatment plant (labeled as F) within the plant premises. Factory-A produces cuptohalosyanine complex (CPC crude), factory-B produces azo pigments, auxiliaries and intermediates, factory-C produces synthetic organic dye stuff and pigments dye stuff, auxiliaries and intermediates, azo and diazo reactive dyes, disperse dyes and intermediates, factory-D produces G Salt and R salt, Factory-E produces acid black, red, blue violet, yellow. F- (WTP) Common effluent treatment plant receives wastewater (primarily treated) from >50 dye producing units, factory G - produces reactive dyes, and factory - H produces tartrazine, indigo carmine, allura red. All of the effluent samples of these factories were stored in amber glass bottles (2.5 L) and transported to the laboratory at the earliest in a lightproof ice chest, and stored under refrigeration (at 4 ° C) in darkness until analysis and bioassay. Dissolved oxygen (DO) (WTW OXI-96 oxy-meter), conductivity (TD Scan conductivity meter) and pH (W T W pH - meter) were also measured as soon as samples arrived to the laboratory. Temperature ( ° C) was recorded at the sampling site.

Daphnia magna Straus used in the experiment were selected from the laboratory stock culture maintained at 25±2 ° C, in 5-litre borosilicate glass beakers and were fed on green algae Selenastrum sps. and yeast powder. The daphnids were reared in photoperiod of 12 h light/12 h dark cycle, with light > 600 Lux (Metcix AVO LM 4 Lux-Meter, France). Reconstituted dilution water (RDW) was used for dilution of effluents to which Daphnia culture was maintained.

To evaluate the aquatic toxicity of dye effluents author performed single species laboratory toxicity test with Daphnia magna, and the 48 h acute toxicity test was conducted after exposing them to various concentrations of the effluent in RDW within 48 h after the effluent sample had arrived in the laboratory. The experimental concentrations were 100, 75.0, 66.6, 50, 33.3, 25, 12.5, 6.25, 3.12, 1.56, 0.8 and 0.4 % of the effluent. Control water and effluent dilutions were prepared with reconstituted dilution water (RDW) with the same formulation used for culture water. Reconstituted water was aerated 12 h before effluent dosage. All the toxicity tests were run at 25±2 ° C. Test chambers were not aerated during experimentation. Twenty-four hours prior to the test adult daphnids (gravid females) were sorted and the young one (neonates) produced from these adults were used in the experiment. Batches of five neonates (≤ 24h) received from gravid females were placed in glass beaker of 100 ml containing 50 ml test volume with two set of control group. Each concentration was replicated a minimum of 4 times. The numbers of immobilized daphnids were recorded after 24 h and 48 h exposure. Daphnids unable to swim for 15 s after gentle stirring were considered as immobile/dead [ ₆ ]. The test protocol is summarized in Table 1, and details of the methodology is described elsewhere [ ₁₉ ]. The experiment was validated with positive control test using reference substance potassium dichromate (K₂Cr₂O₇). The 24 h EC₅₀ was 0.813 (0.70–1.01) mg/L, after ISO [ ₇ ].

Figure 1

Table 1: Summary of test protocol for

The acute toxicity data were analyzed when two or more partial immobilizations were observed. The EC₅₀ and its 95% confidence limits were calculated by probit analysis method to using SPSS software 6.1. The regression analysis was also performed to determine the correlation between x and y variables for regression equation and r ² values. r ² is the correlation coefficient and reflects statistical significance between dependent and independent variables. Toxicity unit (TU) value was calculated using EC₅₀ (100/EC₅₀, expressed as volume percentage). Ratio of acute toxicity was calculated by dividing 48 h EC₅₀ with 24 h EC₅₀.

Results and Discussion

The physico-chemical characteristics of the reconstituted dilution water (RDW) used in the experiment showed that water used in this study had pH 7.8, calcium hardness (as CaCO3) 180.0 mg/L, conductivity 0.7 mS. The physico-chemical characteristics of the effluents differed substantially from one another with respect to chemical characteristics, as expected due to a relatively wide spectrum of dyes manufacturing sources. Dissolved oxygen concentration of the effluents ranged from 3.5–5.2 mg/L, the pH ranged from 7.2–8.0 and conductivity from 0.8–1.5 mS and temperature 26.0-31.0 ° C. Acute toxicity test results of 8 dyes industries (24 and 48 h calculated EC₅₀ and its 95% confidence limit) are given in Table 2. Effluent-A showed highest toxicity causing 100% mortality at 3.13% effluent concentration and more than 50% mortality in 1.5% effluent concentration. The effluent of industry ‘G' showed very low toxicity and was found toxic at the concentration of full strength (100%) causing ≤ 20% mortality in 24 h and ≤ 25% mortality in 48 h. However, the effluent of industry ‘H' had lowest toxicity causing only 10% mortality at 100% effluent. Hence, the EC₅₀ value could not be derived for both effluents. The 24 and 48 h EC₅₀ value for dye industries G and H were always >100, and r ² =0.56.

The 24 h EC₅₀ values and the confidence limit for the effluent of dye industry A, B, C, D, E and F were 1.35 (1.13–1.63), 17.32 (13.94–21.82), 17.67 (14.45–21.61), 29.18 (25.93–33.27), 46.49 (33.43–73.59) and 25.11 (22.55–27.62)%, respectively. The linear regression analysis performed reflected statistical significance between dependent and independent variables (r ² 0.85 – 0 .95). The 48 h EC₅₀ values for effluent of dye industry A, B, C, D, E and F were 1.23 (1.02–1.48), 14.12 (11.53– 17.42), 15.52 (12.68–18.86), 24.53(21.48–28.18), 29.69 (26.54–33.55) and 23.22 (20.70–25.94)%, respectively (Table 2).

Figure 2

Table 2: 24 h and 48 h EC50 values, its 95% confidence limits and ratio, and toxicity units (TU) for dyes industrial effluents

The linear regression analysis performed reflected statistical significance between dependent and independent variables (0.84 < r ² 0.98). The 24 and 48 h EC₅₀ value for the effluent of dye industries G and H were >100%, and r ² = 0.56, showing least toxic response. The regression equations and correlation coefficients were also worked out. The relationship is a sigmoid curve, but can also be expressed in linear fashion with a high degree of confidence (r ² ). The coefficient of determination was as high as r ² = 0.98. Out of 8 effluents tested 5 showed 100% immobility of daphnia at 50% of the effluent concentration, while 1 showed heights toxicity (100% immobility) at 3.13% concentration of the effluent and remaining two-showed lowest toxicity at full strength (100% effluent concentration).

In toxicological experiments, the time of exposure has large effect on biological response. The general rule of thumb is that the longer the exposure time lesser the EC₅₀ value and greater is the toxicity. In our study too, results showed similar pattern having lesser 48 h EC₅₀ value than 24 h EC₅₀ (Table 2). The ratio of EC₅₀ (24 h to 48 h) was found in between 1.1–1.2 except for one sample (1.5), which showed the delayed acute toxic response (Table 2). Kim et al. [ ₂₀ ] reported the acute toxicity ratio (24 h to 48 h) for hexavalent chromium 1.2. Our results are in accordance with the above report.

The classification of effluent [ ₂₁₂₂ ] based on general criteria (Table 3) showed that out of 8 effluent tested one was moderately acutely toxic (48 h EC₅₀ >1.2%), 5 were minor acutely toxic (48 h EC₅₀ >14.12 –29.69%) and the remaining 2 were not acutely toxic (48 h EC₅₀ >100%). Researchers [ ₃₁₄₂₃ ] used the toxicity classification based on Toxicity Unit to classify the effluents toxicity. Based on this classification two effluent sample (G and H) had TU < 1 and remaining 6 were having TU >1.

Figure 3

Table 3: General criteria of toxicity classification and relationship between LC, LD and EC and toxicity rating

Despite the recent developments in chronic toxicity test, traditional acute toxicity (WET) test are still widely used as standard eco-toxicological procedure by environmental agencies/toxicology laboratories to assess the potential impact on ecological system [ ₂₄ ]. Hence the acute toxicity test was conducted exposing the Daphnia magna to treated wastewater of eight dye producing industries. The acute multi-concentration toxicity test yielded a range of response values as a function of effluent concentration, which causes some difficulty in assessing toxicity for effluent classification. However, assessment of industrial effluent by means of acute toxicity test seems to be sufficient for preliminary classification of effluent whether toxic or non toxic. Some authors have already used this idea to assess effluent in terms of their acute toxicity. If the sample whose 48 h EC₅₀ > 100% or show null or poor response of the test organism in a bioassay could be classified as non-toxic [ ₂₁₂₂ ].

Eco-toxicity is the conservation of living communities in receiving aquatic ecosystems, it seems reasonable that those effluents having >1 TU be considered as Toxic, since this would imply that full-strength effluent would adversely affect 50% or more of the exposed organisms. Accordingly, samples having a 48-h EC₅₀ > 100% or which show null or poor response of the test organisms in the bioassays could be classified as Non-Toxic. In accordance with this criterion, only two effluent samples (G and H) in the present study were classified as Non-Toxic. Criteria to classify effluents into toxicity categories (from Low Toxic to Very Toxic) are more difficult. Calleja et al. [ ₂₅ ] have proposed for leachates assessed with Daphnia magna acute toxicity tests the category of Low Toxic when 100 > EC₅₀ > 75%, Toxic for 25 < EC₅₀ < 75%, and Very Toxic for EC₅₀ < 25%. Based on these criteria, it is shown that among the 8 industrial effluents 2 had 48-h 100 >EC₅₀ > 75%, 1 had 25< EC₅₀ 75%, 5 had EC₅₀ < 25%. Thus they can be considered as being Very Toxic. In toxicity units, Very Toxic effluents have >4 TU, only 2 samples (G and H) could be considered to be Low Toxic (TU 1) following the guidelines by Calleja et al. [ ₂₅ ]. Remaining 6 samples are considered to be Toxic >4 TU (25 < EC₅₀ < 75%), i.e. A, B, C, D, E and F.

The results of low exposure concentration range are suitable for determining the no effect concentration (NOEC). The NOEC is the concentration at which a substance does not cause significant effect in comparison with control sample. It has an important ecological implication because it indicates the concentration above which adverse effect can be expressed. The use of ECx values (EC_10/20) for responses <50% has often been chosen in effluent assessment, since these values are usually closer to the acute no-effect concentrations (NOEC). The observed acute no effect concentrations were 0.4, 6.25, 6.25, 12.25, 16.67, 12.25, 66.67 and 75% for effluents A, B, C, D, E, F, G and H, respectively. The EC₁₀ values derived by probit analysis showed closer to acute NOEC. The EC₁₀ values were 0.75, 7.39, 8.59, 14.24, 19.89 and 15.65 for effluent A, B, C, D, E and F.

Toxicological criteria, such as ECx, acute or chronic NOEC, which are used as parameters for effluent toxicity assessment, should be regarded as ‘hypothetical' doses that will not cause adverse effects in the communities of the receiving aquatic systems, since the real environmental effects are subject to abiotic and biotic modifying factors which cannot be precisely predicted [ ₄ ]. However, assessment of industrial effluents by means of acute toxicity tests seems to be sufficient for a preliminary classification of effluents. Some authors have already used this idea to assess effluents in terms of their acute toxicity. Thus, samples whose 48-h EC₅₀ >100% or which show null or poor response of the test-organisms in the bioassays could be classified as Non-Toxic [ ₂₅₂₆ ]. However, when the EC₅₀ >100%, there can be a risk of long-term toxicity which should be evaluated by means of chronic toxicity tests. Therefore, it is important that effluent assessment involves initial screening based on acute tests followed by chronic bioassays. However, NOEC values are dependent on the dilution factor and NOEC is not a statistical estimation like ECx, which implies that the use of limits for this parameter is not appropriate. Chapman [ ₄ ] has argued against the use of NOECs in regulations and they consider the ECx to be a more consistent and reliable parameter, less subjected to inter and intra laboratory variability. It is not the purpose of this paper to discuss possible alternatives to the classic toxicological parameters, but it is interesting to note that this is currently a subject of debate in the scientific community.

A study conducted by Lanciotti et al [ ₁₄ ] showed that textile wastewaters besides containing dyes have considerable amount of metals viz. chromium, copper, lead and zinc leading to toxicity to aquatic organism. Only 3% of the samples showed considerable acute toxicity (EC₅₀ < 100; TU.1) to Daphnia magna. The EC₅₀ of paper mill wastewater containing dyes had value between 50-100%, and EC₅₀ were >25% [ ₂₇ ]. In another study Tonkes et al. [ ₂₂ ] tested the effluent of 10 chemical industry using traditional acute toxicity test with Daphnina magna showed that out of 10 only 6 had acute toxicity < 100% and 4 had >100. These studies also showed that dyes effluents had acute toxic properties, and therefore should be discharged into the aquatic environment after proper treatment. Finally it is concluded that effluents should always be subjected to acute multi-concentration test, yielding ECx values, which are useful for effluent characterization and ranking in the scale of toxicity classes from non-toxic to very toxic either in v/v% value or by using Toxicity Unit (TU). Result of this study showed that it is not possible to speculate on the mode of the toxic action due to difficulties in characterization of the chemical content of the effluent. Therefore, effluent composition should also be characterized. From the ecotoxicological view point single species toxicity provides limited data, hence a battery of test sps. should be included representing different tropic levels. Studies on wastewater effluents indicated that toxicity test have a viable role to play in water quality monitoring. Our results are intended to be basis for further environmental risk assessment.

Acknowledgements