Detoxification of some pharmaceuticals with activated sludge BATCHANZI WILLIAM NJABON Degree project for Master of Science in Ecotoxicology 30 ECTS Department of Plant and Environmental Sciences University of Gothenburg August 2011 y
Summary Pharmaceuticals belonging to the class of non-steroidal anti-inflammatory drugs (NSAID) like Ibuprofen, Ipren, Naproxen, Bamyl and Voltaren occur frequently in fresh water. The aim of this study was to examine the detoxification in water of these compounds. Acute toxicity tests with Daphnia magna were made to study the detoxification with and without additions of activated sludge. The results were tested by ANOVA for effects of detoxification (increase in EC50 upon storage for 3 weeks) and biodegradation (after addition of activated sludge), but no consistent effect upon degradation was found either with or without addition of activated sludge. Only slight differences in acute toxicity to Daphnia were found among the tested pharmaceuticals, and initial EC50s ranged from 50 to 250 mg/l. The toxicity of most tested compounds decreased over time, but the detoxification rate was slow and not affected by addition of activated sludge. Thus, the detoxification of these pharmaceuticals is expected to be slow both in sewage treatment plans and in the environment. 1
Table of contents Summary... 1 Table of contents... 2 1. INTRODUCTION... 3 1. INTRODUCTION... 3 1.1 Route of entry into Waste Water Treatment Plants (WWTPs)... 4 1.2 WWTP treatment processes... 4 1.3 WWTP removal efficiency... 5 1.4 Removal of pharmaceutical during waste water treatment... 6 1.5 Aim of this study... 7 2. MATERIALS and METHODS... 7 2.1 Selection of test compounds... 7 C 8 H 9 O 2... 8 2.2 Test procedures... 9 2.3. Determination of acute toxicity... 9 2.4 Statistical treatment... 9 3. RESULTS... 10 3.1 Immobilisation tests... 10 3.2 Detoxification... 11 4. DISCUSSION... 18 4.1 Acute toxicity... 18 4.2 Detoxification... 18 5. CONCLUSIONS... 19 Acknowledgements... 20 References... 21 ANNEX A. Primary data from toxicity tests with Daphnia magna... 24 2
1. INTRODUCTION Major attention has been paid to the occurrence, fate and effects of heavy metals like e.g. mercury (Friberg et al., 1986), pesticides like DDT (Carson, 1962), and industrial chemicals found in various products like PCBs (Jensen et al., 1969), and more recently of pharmaceuticals and additives from personal care products in Waste Water Treatment Plant (WWTP) sludge and wastewater. In some cases these were suspected to have detrimental effects in the aquatic environment and on human health (Williams, 2003). During the early 1990 s, with the use of more sensitive analytical methods, scientists have discovered low concentrations (ng/l levels) of several pharmaceuticals and personal care products in sewage treatment plant water and sludge and also in surface waters. But so far little is known on their effects on non-target organism (Ericson et al., 2010; Nieto et al., 2010). Major ecotoxicological concern was raised when a more than 95% decrease in the population of the white-back vulture was noted in the 1990 s in Pakistan and India, and this was due to diclofenac, an analgesic/anti-inflammatory drug (Fent et al., 2006a; Gros et al., 2007; Gros et al., 2010; Oaks et al., 2004). Furthermore, the beta-blocker propranolol was found to have affected benthic organism and zooplanktons in north east Spain (Gros et al., 2007). The relationships between fate, transport and environmental effects of micro pollutants have not been well documented (Kolpin et al., 2002). Pharmaceuticals are micro - pollutants in wastewater treatment plants. They are used to cure, prevent or eliminate diseases in animals and humans (Corcoran et al., 2010; Jones et al., 2001). They are designed to resist biodegradation long enough to attain their beneficial effects, and this explains why they are resistant to biodegradation. Our dependency upon animal farming, for an increasing human population, also implies an increased medication used in this sector. Continuous input to the environment of these chemicals can lead to their accumulation in organisms and transfer of effects along natural food-chains. The high diversity of old and new pharmaceutical products can explain their relatively frequent detection in sewage treatment plants, ground water, surface water and in sediments (Corcoran et al., 2010; Fick et al., 2009; Jones et al., 2001; Zhang et al., 2010). IMS Health (2010) has predicted an increase of 5-7% to be spent on pharmaceuticals in 2011 ($880 billion) in comparison with the 4-5% spent in 2010. Worldwide more than US$ 820 billion were spent on pharmaceuticals, with a subsequent annual increase of 1-2% from 2009. Globally the rate of consumption varies with the economic stability of the countries. North America tops the chart as of 2004 with 45%, followed by Europe 13%, Japan 10% and Australia 1% (Corcoran et al., 2010). It has also been found that 100 tons of drugs under prescription were discarded in Germany in 1995, without taken into consideration non-prescribed drugs (Ternes, 1998). For 25 pharmaceutical product studied in United Kingdom, most were consumed at amounts greater than 10 tons, and for Paracetamol, Metformin hydrochloride and 3
Ibuprofen more than 100 tons were consumed (Jones et al., 2002). Pharmaceuticals being sold in developed countries as antibiotics, analgesics, anti-epileptics, β-blockers, β2- sypathomemetics and lipid regulators are among those frequently detected in the aquatic environment (Corcoran et al., 2010; Jones et al., 2001). 1.1 Route of entry into Waste Water Treatment Plants (WWTPs) Pharmaceutical products like antibiotics are administered to both humans and animals, but they have different entry routes in the environment. When taken orally, the active ingredient can resist the metabolic pathway of the non-target site (i.e. liver), until it reaches its target organ in the body (Choong et al., 2006). Depending on the pharmaceutical in question some are excreted as the parent compound (e.g. β-blockers) while others undergo phase I (addition of new and modification of functional group) and phase II metabolism (addition of functional groups such as acetyl, amino, sulfate, glucuronic, glutathione) rendering them excretable without being completely metabolized in the body (William, 2003).These metabolites may react with other products in the WWTP producing other end products, which may be more toxic than the parent compound (Corcoran et al., 2010; Derksen et al., 2004). The increased usage of pharmaceuticals can explain their increasing concentrations in sewage treatment plants, ground water, surface water and sediments (Zhang et al., 2010). Furthermore, out-dated drugs or unused drugs could be washed into sinks and toilets and then entering into WWTPs. 1.2 WWTP treatment processes In the WWTP, pharmaceuticals may undergo biodegradation, remain in suspension, dissolve in water or bind to sewage sludge, depending on their physico-chemical properties such as chemical structure, Henry s constant (most pharmaceutical have low volatility rate) and octanol/water partitioning coefficient (D'Ascenzo et al., 2003; Jones et al., 2002). Most pharmaceuticals have a log K OW 2.5 indicating low sorption potential, which also implies a low removal from activated sludge (Table 1). There are some processes that best characterize organic compound transformation in a waste water treatment plant. They include biotransformation rate in soil, water, sludge, or sediment, photolysis rate, hydrolysis rate, oxidation rate and reduction rate (Williams, 2003; Van Beelen, 2007). Table 1. Sorption rate to activated sludge depends on log K ow (van Beelen, 2007). When a compound absorbs light, thus causing it transformation, it is referred to as photolysis. It could be direct or indirect, when energy transfer occurs by means of another compound. A value for Henry s constant (Hc) of 10-3 atm mol -1 m -3 or more indicates that the substance is highly volatile. 4
Waste water treatment plants (WWTPs) have a central role in water management (Fig. 1), which means most chemicals which are used in society will pass through a WWTP, and can be detected there. Fig.1. Water cycle within the technosphere (after van Beelen, 2007). Waste water treatment plants are designed to treat domestic municipal waste water, sometimes contaminated with industrial waste discharges, by the removal Suspended Solids (SS), Biological Oxygen Demand (BOD), and nutrients (N and P). The wastewater treatment generates sludge, which might contain harmful concentrations of chemicals (More et al., 2010). 1.3 WWTP removal efficiency More than 50 micro pollutants have been found in WWTPs (Carballa et al., 2004). WWTPs are designed to compile with their discharges and they must meet certain regulatory demands within their zone of influence (mixing zone). Their function is not restricted only to accommodate, but also to create environmentally safe waste water and sludge, which can be used for agricultural purposes. The quantitative removal of 5
pharmaceuticals is generally poor, since the designs of WWTPs are based upon removal of nitrogen, phosphorus, particulate matter and BOD (Lindquist, 2003). Nevertheless they do degrade micro pollutants to a certain extent. In recent years also improvements have been made in the WWTPs to handle micro pollutants. In some parts of Europe also tertiary treatment has been added to some WWTPs which include ultra-filtration, flocculation, ozonation, advanced oxidation, or osmosis (Zorita et al., 2009). Agglomeration of suspended particle occurs after metal salts or organic compounds have been added to the wastewater. This gives rise to coagulation and elimination by decantation (Li and Gregory, 1991). But so far little information has been documented on the removal by coagulation-flocculation (Carballa et al., 2005). One process that has been proven to be equally important for fat removal in WWTPs is flotation. This comprise of the fine particles which adhere to the surface of bubbles. Micro pollutants with lipophilic properties are of great concern, since they dissolve in other lipids (Carballa et al., 2005). The removal of polar compounds during the treatment process of the WWTPs have been found to range from 60% to 90%. Data from previous studies have found a removal rate by WWTPs (Carballa et al., 2004) in Germany from 10% to 90%, in Brazil from 12% to 90% and in the USA about 80% was observed. 1.4 Removal of pharmaceutical during waste water treatment Ternes (1998) determined elimination rate in WWTPs based on the influent and effluent concentrations, and studied the importance of plant construction and season. He found reductions of 7-8% for carbamazepine, 81% for acetylsalicylic acid, 96% for propranolol, 99% for salicylic acid, 81% for naproxen, 26% for diclofenac and 51% for bezafibrate. For these chemicals the reduction rate varied among WWTPs. It has been shown that some treatment plants in the USA had up to a 94-100% elimination of Ibuprofen and Naproxen (Ternes, 1998). So even if the WWTPs were not designed to remove complex compounds like pharmaceuticals, they nevertheless did. With the high number of new and evolving micro-pollutants and nano-pollutants, intensified analytical efforts should be directed towards waste water treatment plants. Four major processes accounts for the removal of pharmaceuticals in the activated sludge process. They include air-stripping, photo transformation, biotransformation and sorption. With a relatively low Henry s constant of less than 10-5, and sometimes low aeration, evaporation should be negligible Likewise photo-transformation is negligible, since the sludge prevent the penetration of sunrays (Sipma et al., 2010). Sorption is probably the dominant removal process for pharmaceuticals in a WWTP. It depends on the interaction of the chemical with microorganisms and their aggregation and precipitation. It is defined by the solid-liquid distribution coefficient (K d ), i.e. the ratio between the concentrations of a substance in the solid to that in the aqueous phase, which depends highly on the K ow (Table 1). 6
From an environmental perspective the most important removal process of pharmaceuticals during waste water treatment is by biodegradation. Depending on their physico-chemical structure, the process of elimination becomes complex (Jones et al., 2005). 1.5 Aim of this study The aim of this study was to examine the toxicity of some analgesic pharmaceuticals products and their detoxification (degradation) by activated sludge in the laboratory. Daphnia magna was selected as the test organism for determination of acute toxicity. 2. MATERIALS and METHODS The methods used for treatment of the samples are described below as well as that used for acute toxicity testing with Daphnia, toxicity identification and statistical treatment. 2.1 Selection of test compounds Pharmaceuticals previously detected in WWTPs and commonly used among humans were selected for this study (see Table 2). 7
Table.2. Data for the pharmaceuticals examined in this study (obtained from www.chemspider.com).. Pharmaceutical, active ingredient per pill Chemical formula and CAS number Molecular weight (gmol -1 ) pka Sorption constant K d (L/kg ss) Octanol water partitioning (Log Kow) Bamyl, 500mg acetylsalicylic acid C 18 H 14 CaO 8 200-707-6 Burana, 400mg ibuprofen C 8 H 9 O 2 398 206 4.91 7 3.79 Ipren, 400mg ibuprofen 15687-27-1 C 13 H 18 O 2 15687-27-1 206 4.91 7 3.79 Naproxen 250mg, C 14 H 14 O 3 Voltaren T, 50mg diclofenac 230 4.15 13 3.10 22204-53-1 C 14 H 10 Cl 2 NNaO 2 318 3.1 16 4.51 Though not representative of all pharmaceutical products, analgesic/anti-inflammatory agents are commonly found in household waste systems as they are non-prescribed drugs. Five members of this group were considered as representative of the Swedish market. For this experiment we used analgesics/anti-inflammatory agents, which from previous studies have been detected at high concentrations. This is because they are administered for symptoms and diseases that are frequent, and as such could be bought from the pharmacy without prescription. The pharmaceutical products were bought from the Swedish retail group named APOTEK ET AB. Further information on each product was obtained from the directive of in the package of each pharmaceutical product. This group of pharmaceuticals have a pka range of 4.9-4.1, which indicates high sorption (sorption increases with low ph) (Fent et al., 2006a, b). The initial concentration for each of the pharmaceuticals were achieved by dissolving one pill in 500 ml of test medium, expect for Voltaren T, for which four pills were dissolved in 500 ml test medium. For each stock solution a serial dilution (factor 0.5) was conducted, obtaining 9 different concentrations in a geometric series. 8
2.2 Test procedures Daphnia magna is a commonly used species in ecotoxicology. In the ecosystem it may act both as predator (on algae and bacteria) and as prey (for macro invertebrates and fish). They were obtained from laboratory cultures maintained at test conditions regarding temperature (20 + 2 o C) and test medium.. The test organisms (neonates 0-24 h old) were collected on the day of the test. The test medium was prepared from deionized water (MilliQ) and stock solutions according to ISO (1996). Each pharmaceutical was dissolved in test medium by the use of a magnet stirrer. Then 50 ml of the test solution were pipetted following serial dilution into Petri-dishes of glass (i.d. 10 cm), in which the test organism were exposed to the pharmaceuticals. For each pharmaceutical concentration, the test organisms were exposed to solutions with sludge and without sludge. The experiment was conducted at room temperature (20 + 2 o C) and ambient laboratory light. The experiment lasted 4 weeks, and new Daphnia were added to the same test solutions each week. Immobilization of the Daphnia was recorded after 24 and 48 h. In order to examine the degradation of the pharmaceuticals, the same test sample (chemical in water) was used throughout the test. For samples with sludge, new sludge (2 ml) was added to the sludge sample on each test occasion. All pharmaceuticals had nine different concentrations with a 0.5 dilution factor plus a control. For Alvedon (500mg) and Bamyl (500mg), the concentration range from 1000 mg/l to 3.90 mg/l. For Burana (400mg) and Ipren (400mg), the concentration range was from 800 mg/l to 3.13 mg/l. For Naproxen mylan (250 mg) the concentration range was from 500 mg/l to 1.95 mg/l. For Voltaren T (25 mg) the concentration range was from 200 mg/l to 0.78 mg/l. Besides the two concentration series of each pharmaceutical (with and without activated sludge) a test with the reference toxicant (K 2 Cr 2 O 7 ) was conducted on all test occasions to check the sensitivity of the test organisms (ISO, 1996). 2.3. Determination of acute toxicity In determining the acute toxicity of the pharmaceuticals in accordance with ISO (1996), recording of immobility after 24 and 48 h was recorded. The present study is based on toxicity, so the change of toxicity over times was determined from the 24-hours and 48- hours acute toxicity test (EC50) for D. magna as end point. 2.4 Statistical treatment A computer program was used to determine the EC50s by probit analysis or moving average (Peltier and Weber, 1985). At times, it was difficult to get 95% confidence limits due to steep dose - response relationship by the probit analysis or moving average angle analysis. EC50s in such cases are without confidence limits. 9
ANOVAs were used to determine the statistical significance of chemical compound, time (detoxification rate) and biodegradation (addition of sewage sludge) on toxicity (24- and 48-h EC50). The same software program (Crunch software corp., Oakland, CA USA) that was used for the ANOVAs was used for derivation of Spearman rank correlation coefficients. 3. RESULTS 3.1 Immobilisation tests In the reference tests, the 24-h EC50 for potassium dichromate, did not differ significantly from the reference interval (0.6 1.7 mgl -1 ), established by the ISO (1996). Fig 1 shows 24-h EC 50 for all pharmaceuticals with sludge ranging 71 mg/l 707 mg/l and without sludge ranging 71 mg/l 354 mg/l, and for the 48-h EC50 ranging 62 mg/l 657 mg/l with sludge and 51 mg/l -354 mg/l without sludge. Fig 1. Daphnia EC50s for the 5 tested chemicals after 24 h exposure (1 a) and 48 h exposure (1b) 1a) 24 h 1b) 48 h 10
3.2 Detoxification The ANOVA shown in table 3 were derived for 24-h as well as 48-h EC50s. This table also gives us statistical significant relationship (p<0.05) for the various parameters, for which only compounds had statistical significance. There was no overall effect on detoxification rate (week), sludge addition or their 2-factorial interactions (CW, CS, WS). Table 3. ANOVA for 24-h and 48-h EC50s After 24 hours Variance component DF SS MSS F P Overall 39 460227.9 Compound (C) 4 232863.7 58215.93 7.865 0.0024 Week (W) 3 29600.1 9866.7 1.333 0.3097 Sludge (S) 1 3062.5 3062.5 0.414 0.5322 CW 12 67701.2 5641.767 0.762 0.6772 CS 4 18016.3 4504.075 0.608 0.6643 WS 3 20161.3 6720.433 0.908 0.466 Error 1 12 88823 7401.917 11
After 48 hours Variance component DF SS MSS F P Overall 39 460227.9 Compound (C) 4 232863.7 58215.93 7.865 0.0024 Week (W) 3 29600.1 9866.7 1.333 0.3097 Sludge (S) 1 3062.5 3062.5 0.414 0.5322 CW 12 67701.2 5641.767 0.762 0.6772 CS 4 18016.3 4504.075 0.608 0.6643 WS 3 20161.3 6720.433 0.908 0.466 Error 1 12 88823 7401.917 Detoxification curves (EC50 vs. time) for the five pharmaceuticals are shown below. In summary most of the pharmaceutical product, show some degree of detoxification after the addition of activated sludge with time. The figures below show the relationship for the different compounds. A slight detoxification was found after 2-3 weeks. 12
The detoxification pattern was similar with and without addition of sludge. The detoxification was higher with addition of sludge at the end of the test period based on 24-h EC50s. 13
Based upon 48-h EC50s there was no general difference in detoxification with and without sludge. Based upon 24-h EC50s the degradation rate seemed higher without sludge. 14
Based on 48-h EC50s there was no major detoxification either with or without sludge. Samples with sludge were consistently more toxic than those without.based on 24-h EC50s There was a slight detoxification both with and without addition of sludge. 15
Also for the 48-h EC50s there was som detoxification, especially without addition of sludge.. A detoxification was found for the 24-h EC50s. 16
The 48-h EC50s showed the same pattern as the 24-h EC50s. Table 4. 24-h and 48-h EC50s, after 0-3 weeks of degradation with sludge(s) and without addition of activated sludge. 24h Week 0 1 2 3 Average Standard Deviation Compound naproxen s 88 94 95 146 106 27, naproxen 123 125 177 155 145 26 burana s 80 76 124 249 132 81 burana 81 85 117 124 102 22 voltaren s 84 88 76 80 82 5 voltaren 71 78 109 100 90 18 ipren s 76 71 109 85 85 17 ipren 92 93 141 141 117 28 bamyl s 177 136 707 264 321 263 bamyl 177 146 177 354 214 95 17
48 h Week 0 1 2 3 Average Standard deviation Compound naproxen s 44 78 73 44 60 18 naproxen 51 73 88 73 71 15 burana s 71 46 70 76 66 13 burana 33 66 66 71 59 17 voltaren s 59 62 66 71 65 5 voltaren 65 64 62 71 66 4 ipren s 71 85 71 70 74 7 ipren 42 67 109 109 82 33 bamyl s 177 136 657 264 309 238 bamyl 146 164 164 354 207 98 4. DISCUSSION 4.1 Acute toxicity Mortality was determined for D. magna when exposed to the five pharmaceutical products. The result indicated about the same acute toxicity of all pharmaceuticals for D. magna. Generally Classification for very toxic compounds to aquatic organism should have values less than 1 mg/l, for toxic compounds it ranges between 1-10 mg/l and harmful from between 10-100mg/l (CEC,1996). The investigated pharmaceuticals can be classified as harmful based on their acute toxicity to D. magna. 4.2 Detoxification The detoxification/degradation rates were generally low and insignificant based on the ANOVAs both with and without addition of activated sludge. From this study, Bamyl had the highest detoxification rate, which is consistent with the 81% degradation rate reported by (Daughton and Ternes, 1999). The EC50 values for 24 h and 48 h were about the same, which facilitates the interpretation. Concentrations of up to 340 ng/l has been found for this chemical in surface water, while those for some river have been 37 ng/l, 36 ng/l and 28 ng/l (Moldovan, 2006).These concentrations are far below those used that were acutely toxic in this study, and the findings on detoxification also indicate less environmental concern for this compound (acetyl salicylic acid). 18
5. CONCLUSIONS The acute toxicity tests made with Daphnia magna on 6 pharmaceutical products (Alvedon, Bamyl, Burana, Ipren, Naproxen Mylan and Voltaren), which are used as analgesic/anti-inflammatory agents, and which are commonly found in household waste systems, have shown that All of them were acutely toxic between 50 and 700 mg/l, making them classified as Hazardous for the environment. Detoxification (abiotic and biotic) studied for 3 weeks was insignificant for most of them, which suggest that they are not easily degraded/detoxified in sewage treatment plants or the aquatic environment. Among these compounds Bamyl (acetyl salicylic acid) showed some detoxification, which is consistent with previous findings on sewage sludge works removal. Altogether the present study has shown that the studied pharmaceuticals do not detoxify/degrade easily and they will therefore most likely behave like persistent chemicals in the environment. 19
Acknowledgements I would like to give special thanks to my supervisor, Göran Dave for his enormous assistance, and his availability despite his health issues. Thanks to the entire stuff at department of Ecotoxicology, for the opportunity and facilities given to me during my two years study period at the Goteborg University. Lastly thanks, to all my mates who contributed in one way or the other during this period. 20
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ANNEX A. Primary data from toxicity tests with Daphnia magna All test results are shown as mobile (living) Daphnia of the 10 added initially after waious times (24 and 48 h) and treatments (with and without addition of activated sludge) WEEK 1 BURANA 400mg Conc./survival 800mg 400mg 200mg 100 50 25 12.5 6.25 3.125 control Sludge24hr 0 0 0 2 10 10 10 10 10 10 Sludge48hr 0 0 0 0 10 10 10 10 10 10 Without24hr 0 0 0 4 9 9 9 10 10 10 Without48hr 0 0 0 0 5 4 10 10 10 10 Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 6 10 10 10 48hr 0 0 5 5 7 10 VOLTAREN T 50mg Conc./survival 200mg 100mg 50mg 25 12.5 6.25 3.125 1.56 0.78 control Sludge24hr 0 7 9 10 10 10 10 10 10 10 Sludge48hr 0 5 8 10 10 10 10 10 10 10 Without24hr 0 7 10 9 10 10 10 10 10 10 Without48hr 0 1 9 9 10 10 10 10 10 10 IPREN 400mg Conc./survival 800mg 400mg 200mg 100 50 25 12.5 6.25 3.13 control Sludge24hr 0 0 0 2 9 10 10 10 10 10 Sludge48hr 0 0 0 0 10 10 10 10 10 10 Without24hr 0 0 0 6 9 9 10 10 10 10 Without48hr 0 0 0 0 4 9 10 10 10 10 24
Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 4 8 10 10 48hr 0 0 0 5 10 10 NAPROXEN MYLAN 250mg Conc./survival 500mg 250mg 125mg 62.5mg 31.3mg 15.6mg 7.8mg 3.9mg 1.95mg control Sludge24hr 0 0 4 6 10 10 10 10 10 10 Sludge48hr 0 0 0 2 8 10 10 10 10 8 Without24hr 0 0 7 9 10 10 10 10 10 10 Without48hr 0 0 0 4 9 9 10 10 10 10 BAMYL 500mg Conc./survival 1000mg 500mg 250mg 125mg 62.5mg 31.3mg 15.6mg 7.8mg 3.9mg control Sludge24hr 0 0 0 10 10 10 10 10 10 10 Sludge48hr 0 0 0 10 10 10 10 10 10 10 Without24hr 0 0 0 10 10 10 10 10 10 10 Without48hr 0 0 0 9 10 10 10 10 10 8 Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 3 8 10 10 48hr 0 0 0 3 10 8 WEEK 2 BURANA 400mg Conc./survival 800mg 400mg 200mg 100 50 25 12.5 6.25 3.125 control Sludge24hr 0 0 0 1 10 10 10 10 10 10 Sludge48hr 0 0 0 0 7 8 10 10 9 10 Without24hr 0 0 0 3 10 10 10 10 10 10 Without48hr 0 0 0 0 9 10 9 10 10 10 25
Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 6 10 10 10 48hr 0 0 5 5 7 10 VOLTAREN T 50mg Conc./survival 200mg 100mg 50mg 25 12.5 6.25 3.125 1.56 0.78 control Sludge24hr 0 4 9 10 10 10 10 10 10 10 Sludge48hr 0 0 7 10 10 10 10 10 10 10 Without24hr 0 6 10 9 10 10 10 10 10 10 Without48hr 0 0 9 9 9 10 10 7 10 10 IPREN 400mg Conc./survival 800mg 400mg 200mg 100 50 25 12.5 6.25 3.13 control Sludge24hr 0 0 0 3 10 10 10 10 10 10 Sludge48hr 0 0 0 1 9 10 10 10 10 10 Without24hr 0 0 0 6 9 10 10 10 10 10 Without48hr 0 0 0 2 7 10 9 7 10 10 Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 6 10 10 10 48hr 0 0 1 1 10 9 NAPROXEN MYLAN 250mg Conc./survival 500mg 250mg 125mg 62.5mg 31.3mg 15.6mg 7.8mg 3.9mg 1.95mg control Sludge24hr 0 0 2 9 10 10 9 10 10 10 Sludge48hr 0 0 1 7 10 10 10 10 10 8 Without24hr 0 0 5 10 10 10 10 10 10 10 Without48hr 0 0 0 7 10 10 10 10 10 10 26
BAMYL 500mg Conc./survival 1000mg 500mg 250mg 125mg 62.5mg 31.3mg 15.6mg 7.8mg 3.9mg control Sludge24hr 0 0 0 6 10 10 10 10 10 10 Sludge48hr 0 0 0 6 10 10 10 10 10 10 Without24hr 0 0 0 9 10 10 10 10 10 10 Without48hr 0 0 0 9 10 10 10 10 10 8 Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 3 10 10 10 48hr 0 0 0 3 7 8 WEEK 3 BURANA 400mg Conc./survival 800mg 400mg 200mg 100 50 25 12.5 6.25 3.125 control Sludge24hr 0 0 0 8 10 10 10 10 10 10 Sludge48hr 0 0 0 0 10 6 10 10 10 4 Without24hr 0 0 0 7 10 10 10 10 10 10 Without48hr 0 0 0 0 9 10 10 10 10 4 Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 9 10 10 10 48hr 0 0 0 2 10 10 VOLTAREN T 50mg Conc./survival 200mg 100mg 50mg 25 12.5 6.25 3.125 1.56 0.78 control Sludge24hr 0 0 0 8 10 10 10 10 10 10 Sludge48hr 0 0 0 0 10 6 10 10 10 4 Without24hr 0 6 10 10 10 10 10 10 10 10 27
Without48hr 0 0 8 9 9 10 10 10 10 4 IPREN 400mg Conc./survival 800mg 400mg 200mg 100 50 25 12.5 6.25 3.13 control Sludge24hr 0 0 0 6 10 10 10 10 10 10 Sludge48hr 0 0 0 0 10 10 10 10 10 10 Without24hr 0 0 0 10 10 10 10 10 10 10 Without48hr 0 0 0 6 9 10 10 10 10 10 Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 8 10 10 10 48hr 0 0 0 3 10 10 NAPROXEN 250mg Conc./survival 500mg 250mg 125mg 62.5mg 31.3mg 15.6mg 7.8mg 3.9mg 1.95mg control Sludge24hr 0 0 3 8 10 10 10 10 9 10 Sludge48hr 0 0 0 7 10 10 10 10 9 10 Without24hr 0 0 10 10 10 10 10 10 10 10 Without48hr 0 0 1 9 10 10 10 10 10 10 BAMYL 500mg Conc./survival 1000mg 500mg 250mg 125mg 62.5mg 31.3mg 15.6mg 7.8mg 3.9mg control Sludge24hr 0 10 10 10 10 10 10 10 10 10 Sludge48hr 0 9 9 9 10 10 9 9 10 10 Without24hr 0 0 0 10 10 10 10 10 10 10 Without48hr 0 0 0 9 9 10 10 10 10 9 Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 10 8 10 10 48hr 0 0 4 10 10 10 28
WEEK 4 BURANA 400mg Conc./survival 800mg 400mg 200mg 100 50 25 12.5 6.25 3.125 control Sludge24hr 0 0 8 10 10 10 10 10 10 10 Sludge48hr 0 0 0 1 10 10 10 10 10 10 Without24hr 0 0 0 8 10 10 10 10 10 10 Without48hr 0 0 0 0 10 10 10 10 10 10 Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 10 10 10 10 48hr 0 0 0 6 10 10 VOLTAREN T 50mg Conc./survival 200mg 100mg 50mg 25 12.5 6.25 3.125 1.56 0.78 control Sludge24hr 0 2 10 10 10 10 10 10 10 10 Sludge48hr 0 0 10 10 10 10 10 10 10 10 Without24hr 0 5 10 10 10 10 10 10 10 10 Without48hr 0 0 10 10 10 10 10 10 10 10 IPREN 400mg Conc./survival 800mg 400mg 200mg 100 50 25 12.5 6.25 3.13 control Sludge24hr 0 0 1 3 10 10 10 10 10 10 Sludge48hr 0 0 0 0 10 10 10 10 10 10 Without24hr 0 0 0 10 10 10 10 10 10 10 Without48hr 0 0 0 6 8 10 10 10 10 10 29
Positive Control (potassium dichromate) K 2 Cr 2 O 7 Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 1 8 10 10 10 48hr 0 0 0 5 10 10 NAPROXEN 250mg Conc./survival 500mg 250mg 125mg 62.5mg 31.3mg 15.6mg 7.8mg 3.9mg 1.95mg control Sludge24hr 0 0 7 9 10 10 10 10 10 10 Sludge48hr 0 0 0 0 10 10 10 10 10 10 Without24hr 0 0 8 10 10 10 10 10 10 10 Without48hr 0 0 0 7 9 10 10 10 9 10 BAMYL 500mg Conc./survival 1000mg 500mg 250mg 125mg 62.5mg 31.3mg 15.6mg 7.8mg 3.9mg control Sludge24hr 0 9 10 10 10 10 10 10 10 10 Sludge48hr 0 9 10 10 10 10 10 10 10 10 Without24hr 0 0 10 10 10 10 10 10 10 10 Without48hr 0 0 10 10 10 10 10 10 10 10 Positive control (potassium dichromate) Conc./ 4mg 2mg 1mg 0.5mg 0.25mg control survival 24hr 0 0 5 10 10 10 48hr 0 0 1 7 10 10 30