whole effluent toxicity assessment of industrial effluents

Journal of Water and Environment Technology, Vol.12, No.1, 2014

Address correspondence to Takashi Kusui, Department of Environmental Engineering, Toyama Prefectural University, Email: [email protected] Received May 10, 2013, Accepted September 12, 2013.

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Whole Effluent Toxicity Assessment of Industrial Effluents in Toyama, Japan with a Battery of Short-term Chronic Bioassays Takashi KUSUI*, Yuri TAKATA*, Yasuyuki ITATSU*, Jinmiao ZHA**

*Department of Environmental Engineering, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan

**Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, Haidian District, Beijing 100085, China

ABSTRACT Eight industrial effluents from Toyama Prefecture, Japan were subjected to three freshwater short-term chronic assays to analyze the potential of ecotoxic impact on receiving water. Tests comprised algal inhibition (Pseudokirchneriella subcapitata), crustacean reproduction (Ceriodaphnia dubia), fish embryo-larva (Danio rerio). Among the eight effluents, chronic toxic effects on alga, crustacea and fish were observed in six, six and one effluents respectively. No-observed-effect concentrations (NOECs) equal to or less than 5% were found in five samples. Nickel and salts were suspected as the causative factors from comparing water parameters with their effects. The impact of these effluents after discharged into receiving water is discussed. The results of the study show the effectiveness of the whole effluent toxicity approach to compensate the present shortcoming of wastewater regulation in Japan. Keywords: aquatic organism, ecotoxic effect, industrial effluent, whole effluent toxicity (WET)

INTRODUCTION Direct discharge of industrial effluent into aquatic ecosystems continues to be an important area of concern because of the potentially ecotoxic impact on receiving water biota. Due to the usually unknown, complex, and often highly variable composition of effluents, it is very difficult to predict and control their effects on aquatic ecosystems based on measured chemical parameters in effluent standards. In order to compensate the shortcomings of traditional effluent regulation, whole effluent toxicity (WET) testing has been introduced in the USA, Canada, European countries and South Korea (USEPA, 1991; Kusui, 2000; Tatarazako, 2006). The Japanese Environmental Ministry established an advisory body in 2009 to examine the feasibility of introducing the WET approach into the present regulatory framework. In March, 2013, draft guidelines for WET testing were released, which proposed three freshwater short-term chronic tests (algae, crustacea and fish). However, there are few reports on WET testing of industrial wastewater in Japan (Kusui and Blaise, 1999; Yamamoto et al., 2010) and there is an urgent need to expand knowledge of WET testing and grasp the ecotoxic potential of present industrial effluents before introducing the new approach. This work presents data from three proposed short-term chronic assays of eight industrial effluents collected in Toyama, Japan. Causative factors attributed to the apparent toxicity were estimated from measured water parameters. The potential effects


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of effluents on aquatic ecosystem were discussed in relation to the dilution factors in receiving water bodies. MATERIALS AND METHODS Sampling and preparation of industrial effluents Between October, 2012 and January, 2013, grab samples of final effluents were collected from eight industrial sites located in Toyama, Japan. These sites are designated as specified facilities under Water Pollution Control Law and all effluents met the criteria of present effluent standards. At each site, final effluent was collected in glass bottles and immediately transferred to the laboratory in coolers with ice packs. In preparation for chemical analyses and bioassays, samples were filtered through a glass fiber filter (GA-55, Advantec, Japan), then, stored in glass bottles at 4°C until testing. In principle, biological testing was conducted within 36 h of sampling. Analysis of water parameters Among the water parameters, pH and DO were measured on-site by pH meter (pH/COND METER D-54, HORIBA, Japan) and DO meter (ProODO, YSI, USA), respectively. In the laboratory, pH and electrical conductivity (EC) were measured by pH meter (HM-30R, TOADKK, Japan) and pH/EC meter (pH/COND METER D-54, HORIBA) respectively. Hardness was measured according to standard methods (Japan Water Works Association, 2011). Both free and total chlorine were measured by spectrometer (DR2800, HACH, USA). Since effluent #5 contained high levels of residual chlorine, it was dechlorinated by sodium thiosulfate before biological tests. BOD, T-P and T-N were measured according to JIS K0102 (Japanese Standards Association, 2010). Metals in the effluents were determined as follows: 50 mL of filtered effluent was mixed with 5 mL nitric acid (EL grade, Kantokagaku, Japan) in a metal-free PP tube (DigiTUBEs, SCP Science, Canada). The mixture was digested on a hot-plate (DigPREP, SCP Science) by wet digestion method. The digested sample was made up to 50 mL by adding Milli-Q water, and then analyzed by ICP-MS (Agilent 7700e, Agilent Technologies, USA). Whole effluent toxicity tests The suite of three short-term chronic bioassays chosen for this study represents three trophic levels (primary producers, primary/secondary consumers) and are proposed in the draft Japanese WET testing guidelines. In each bioassay, organisms were exposed to effluents at a series of dilutions (0, 5, 10, 20, 40 and 80%). Some samples with high toxicity were diluted to less than 5%. The algal growth inhibition test was carried out with the green microalgae Pseudokirchneriella subcapitata (NIES-35) in accordance with OECD test guideline TG201 (OECD, 2006). Algal suspensions (60 mL) inoculated at 0.5 × 104 cells/mL in an Erlenmeyer flask (200 mL capacity) were exposed to a range of effluent concentrations prepared with AAP medium. Samples were prepared in triplicate for each effluent concentration. The samples were incubated under continuous illumination from fluorescent lamps (ca. 60 μmol/m2/s) at a temperature of 23 ± 2°C in


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an orbital shaking culture. Algal cell density was determined with a particle counter (detection range 3 – 12 μm, CDA-500, Sysmex, Japan) every 24 h in the 72-h growth test. EC50 and NOEC for growth rate were determined with analysis software (Ecotox-Statics ver. 2.6, The Japanese Society of Environmental Toxicology, Japan). Cladoceran reproduction tests were assessed via a three-brood renewal toxicity test with Ceriodaphnia dubia standardized by Environment Canada (Environment Canada, 2007). At the beginning of the test, one neonate daphnid (< 24 h old) was transferred to a glass containing 15 mL of diluted samples. Each treatment consisted of 10 replicates of a particular test concentration or the control. During the test, the samples were incubated under illumination (light 16 h/ dark 8 h) at a temperature of 25 ± 2°C. Appropriate volumes of food (YCT and algae) were added daily and each test solution was renewed three times per week. The death of first-generation daphnia and the number of live neonates produced by first-generation daphnid were observed for 8 days. EC50 and NOEC for fecundity were determined with analysis software (Ecotox-Statics ver. 2.6). Short-term toxicity test on fish sac-fry stages was conducted according to OECD TG212 (OECD, 1998). Briefly, 10 fertilized eggs (< 4 h) were placed in a glass containing 50 mL of test solution. Each treatment comprised 4 replicates of a particular test concentration or the control. During the test, embryos were incubated under illumination (light 16 h/ dark 8 h) at a temperature of 26 ± 1°C. Hatching and survival rates were observed daily during the test period (10 days). Based on survival and hatching rates, NOEC was calculated with analysis software (Excel Tokei ver. 6.0, Esumi Inc., Japan). To compare the results of toxicity tests, chronic toxicity units (TUc) were calculated with the following formula:

TUc = 100/NOEC (%) (1) RESULTS AND DISCUSSION Effect on aquatic organisms Figure 1 shows the concentration–effect relationship on algal growth. Effluents #2, #4, #5, #6 and #7 showed stronger adverse effect with increase in concentration. The NOEC ranged from 5% to 20% among these samples. For effluent #1, slight inhibition was observed only at the highest concentration (i.e. 80%). In contrast, effluents #3 and #8 did not show any inhibition. In these cases, NOEC was designated as 80%, which was the highest test concentration. Figure 2 shows the concentration–effect relationship on reproduction and survival of C. dubia. Five effluents showed strong effect. Mortality within 48 h was observed in effluents #2, #5 and #6. In contrast, mortality occurred after 48 h exposure for effluent #1, #3 and #4. This delayed effect may suggest slow-acting causative factor for effluent #1. For effluents #4 and #5, the organisms were exposed to lower concentrations, from 0.625% to 5%. However, reproduction was totally inhibited at the lowest concentration (0.625%) for effluent #6. Although effluent #5 showed strong effect on reproduction at the lowest concentration (5%), an additional test was not possible owing to time


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constraints. Effluents #7 and #8 showed no significant concentration–effect relationship, and therefore, NOEC was estimated at 80%.

Fig. 1 - Effect on algae. Error bars represent the standard error (** p < 0.05, * p < 0.01)

from the mean (control: n = 6, treatment: n = 3).

Fig. 2 - Effect on daphnia. Error bars represent the standard error (** p < 0.05, * p < 0.01) from the mean (n = 10).


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Figure 3 shows the concentration–effect relationship on survival and hatching of fish. In contrast to the results of other tests, only effluent #2 showed toxicity. Although the hatching rate was reduced significantly to 79.3% at the concentration of 80%, no larvae survived at the end of the test. The sudden death after hatching can be explained by the loss of barrier provided by the chorion membrane (Ensenbach and Nagel, 1995).

Summary of chronic toxicity of effluents Figure 4 shows the TUc of each effluents. Of the eight effluents studied, only one did not show any toxicity to aquatic organisms. Five of the effluents had relatively strong toxicity, defined as TUc of 20 or more. The number of effluents that showed chronic effect on algae, crustacea or fish was 6, 6, and 1, respectively. Although algae and daphnia were both susceptible to six of the eight effluents, crustacean reproduction was more sensitive than algal inhibition in terms of TUc. The highest TUc was 160, indicating that NOEC was as low as 0.625%.

Fig. 3 - Effect on fish. Error bars represent the standard error (** p < 0.05, * p < 0.01) from the mean (n = 4).


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Fig. 4 - TUc of each effluent. Estimation of causative factors Table 1 summarizes the water parameter analysis, highlighting several characteristic findings. Firstly, effluent #2 showed relatively high electrical conductivity (EC), indicating higher electrolyte contents. Secondly, effluent #5 contained total and combined residual chlorine as high as ca. 1 mg/L, although the sample was collected at a point where it was not affected by disinfection treatment. Thirdly, effluents #1, #4, and #6 showed relatively high concentrations of Ni, which was reported to be relatively toxic to aquatic organisms. In a previous study, 96-h NOEC for alga (Selenastrum subcapitata) was reported as 10 μg/L (Chao and Chen, 2000). NOEC of nickel for reproduction of C. dubia ranged from < 3.8 μg/L to 5.8 μg/L depending on water hardness (Keithly et al., 2004). For the effect on early life stage of zebrafish, no effect concentration for survival was estimated to be 80 μg/L (Dave and Xiu, 1991). Therefore, it is concluded that the higher TUc of algae and daphnia for effluents #1, #4, and #6 was caused by Ni toxicity. According to PRTR data, as of 2002, Toyama Prefecture had the second highest Ni discharge to the aquatic environment in Japan, and alumite electrolytic coloring process was suspected as the source of Ni discharge (Research Center for Chemical Risk Management and National Institute of Advanced Science and Technology, 2008). The toxicity of effluent #2 with high EC (5.23 S/m) might be partly explained by high concentration of electrolyte. In our study, algal NOEC of NaCl was 2,400 mg/L. At this concentration, the solution has EC of 1.86 S/m, which corresponds to 35% the EC of effluent #2. Additionally, NOEC of NaCl for reproduction of C. dubia was 670 mg/L in our study. Therefore, it is suspected that high concentration of salts produced the inhibitory effect on algal growth and daphnia reproduction. For effluent #5, it is difficult to estimate the causative factor from measured parameters. One possibility is that residual chlorine which was suspected to be a by-product of manufacturing process, remained after dechlorination as a result of the slow-reacting character of combined chlorine. For acute toxicity of combined residual chlorine on C. dubia, 24-h LC50 of monochloramine and dichloramine was 0.016 and 0.027 mg/L, respectively (Taylor, 1993) although no data was available for acute toxicity on algae.


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Table 1 - Results of water analysis.

The discussion above was only an estimation based on a limited number of measured parameters and might miss the contribution of factors not measured. To identify and confirm the cause of toxicity observed in this study, a further toxicity identification evaluation (TIE) is necessary. Potential effect on aquatic ecosystems The potential impact of industrial discharges on the aquatic ecosystem was evaluated based on TUc’ after dilution by receiving water bodies, as follows:

TUc’ = TUc/dilution factor (2) For the chronic criteria, USEPA proposed 1.0 TUc at the end of the mixing zone to prevent any chronic toxicity in receiving water outside the mixing zone (USEPA, 1991). In Japan, there are no environmental criteria or standards based on units of toxicity, therefore, TUc of 1.0 was tentatively used in this study. To calculate TUc’, the dilution factor was determined in two ways. In Case 1, dilution factor was designated as 10, based on the rationale that most Japanese effluent standards are set to ten times the environmental standards of water bodies. In Case 2, dilution factor was calculated in the low-flow condition in water bodies. Flow rate exceeds the low-flow on 275 days per year. The reason for using this factor is that critical condition can occur during low-flow period. To calculate this factor, the low-flow of receiving rivers was divided by the discharge of industrial effluent. The dilution factor ranged from 0.30 to 1,810 in Case 2. However, effluent #6 was discharged into sea, and therefore no dilution factor was calculated. Figure 5 shows TUc’ in Case 1. Five effluents exceed the criteria, whereas only two effluents exceed the criteria in Case 2 (Fig. 6). As shown in Case 1 when 10-fold dilution was applied, it is likely to overestimate the environmental impact, but it will be on the safe side for environmental protection. However, it is also clear that influence may be underestimated for effluent #4 with a dilution factor of 0.3. Thus further study is required to compare the WET measurements with impacts on receiving water and to provide a rationale for setting an appropriate dilution factor.


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Fig. 5 - TUc’ in Case 1. Fig. 6 - TUc’ in Case 2.

CONCLUSIONS Eight industrial effluents from locations in Toyama Prefecture, Japan were subjected to three freshwater short-term chronic assays. Of the eight effluents, chronic toxic effect on algae, crustacea, and fish was observed in six, six, and one effluent, respectively. Nickel and salts were suspected to causative toxicity in some effluents. Some effluents were suspected to have toxic effect even after dilution by receiving water. The results show the potential effectiveness of the WET approach in improving wastewater regulation and protecting aquatic environments in Japan. ACKNOWLEDGEMENTS The authors thank Mr. Atsushi Sawai and Dr. Nobukazu Miyamoto (IDEA Consultants, Inc.) for their technical advice in conducting WET tests; the staff of Toyama Prefectural Environmental Science Research Center for analyzing BOD, CODMn. Greater Nagoya environmental field researchers from the oversea invitation program grant gave us the opportunity to conduct this work with bilateral collaboration. REFERENCES Chao M. R. and Chen C. Y. (2000) No-observed-effect concentrations in batch and

continuous algal toxicity tests. Environ. Toxicol. Chem., 19(6), 1589-1596. Dave G. and Xiu R. (1991) Toxicity of mercury, copper, nickel, lead, and cobalt to

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whole effluent toxicity assessment of industrial effluents

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