Ecotoxicology Assessment of Waste Water Emitted from Radomir Metal Industries (Bulgaria)

ECOLOGIA BALKANICA 2012, Vol. 4, Issue 2 December 2012 pp. 51-60

Ecotoxicology Assessment of Waste Water Emitted From Radomir Metal Industries (Bulgaria)

Мariana I. Lyubenova*1, Snejana B. Dineva2, Irina B. Karadjova3

1 - Department Ecology and EP, University of Sofia, Faculty of Biology, 8 D. Tzankov Blvd., 1164 Sofia, BULGARIA 2 - Technical College of Yambol, University of St. Zagora, Gr.Ignatiev Str. 38, 8602Yambol, BULGARIA 3 - Department of General and Inorganic Chemistry, University of Sofia, Faculty of Chemistry, 1 J. Bourchier Blvd., 1164 Sofia, BULGARIA * Corresponding author: [email protected]

Abstract. The purpose of this article is to evaluate the toxicological effect of wastewater emitted from „Radomir Metal Industries". It has been registered that the range of 50% mortality (LC50) of great water flea (Daphnia magna St.) is limited between 75% and 80% effluent. The data mortality rate-effluent dilution for Pseudorasbora parva well correlated with linear regression, R2=0.86. LC50 is reported from exposure to raw sewage (100%). Results indicate that even when individual concentrations of toxic metals are within the permissible limits the effluent remains toxic for the hydrobiota perhaps due to the combine effect of the contaminants.

Keywords: ecotoxicology, heavy metals, effluent, D. magna St., P. parva Temminck & Schlegels.

Introduction aquatic systems. However, it is reckoned Metals are perceived as pollutants but that such chemical mixtures where indivi- they are also naturally existing substances in dual constituents are present at low, non- the environment. Elements like Cd, Hg, Pb, toxic concentrations may trigger toxicity due Ni are accepted as priority pollutants for to additive or synergistic effects among the aquatic environment. Most metals do not constituents (RAJAPAKSE et al., 2002; undergo microbial or chemical degradation MONTVYDIENE & MARCIULIONIENE, 2004). and are toxic and their total concentration in The surface water environment is under soils persists for a long time after their the influence of continuous industrial introduction (ADRIANO, 2003; KIRPICHTCH- pressure. It has long been recognized that IKOVA et al., 2006). Their content in aquatic the formation of organic and inorganic metal environment is very divers, and it was well complexes and metal sorption to particulate known that neither total nor dissolved total material reduces metal bioavailability and metal concentrations are good predictors for toxicity in the water compartment (LUCK et their bioavailability and toxicity (NEWMAN, al., 2008; SAEEDI et al., 2004). Bio-assessment 2008). Many chemical mixtures, with con- can be used as a tool to detect the presence centrations of individual chemicals consi- of hazardous chemicals in the environment dered as nontoxic, are often presented in evaluating the effects of mixtures with the

Ecotoxicology Assessment of Waste Water Emitted From Radomir Metal Industries (Bulgaria) combined effects that can be expressed as facilities fully meet the requirements of the synergism, additivity and antagonism European Standards for the quality of the air demonstrating bioavailability of and water (http://radomirmetal contaminants to different species (LOUREIRO industries.com/en/application/index). et al., 2006; LANDIS et al., 2011; LYUBENOVA & „Radomir Metal Industries" has a KALCHEV, 2011; LYUBENOVA et al., 2012). complex permit № 145-NO/2008 on within However assessment of water quality in the the scope of paragraph 2.2. Annex 4 of the presence of mixture of pollutants by using Law on Environmental Protection. Under single–species biotest may be insufficient for the complex permit "Radomir Metal a more biologically complex system; there- Industries has established annual emission fore, organisms of different phylogenetic standards presented in Table 1. levels and ontogenesis have to be involved It was reported (PASKALEV, 2001), that in these investigations (MONTVYDIENE & there are some strongly polluted sections in MARCIULIONIENE, 2004). Struma River – after the towns of Pernik, The purpose of article is to evaluate the Kjustendil, and Dupnitza. Sources of toxicological effect of wastewater emanated pollution are the industrial wastewater of from Radomir Metal Industries". the towns of Radomir and Pernik, as well as domestic sewage of some of the bigger Material and Methods settlements (the town of Blagoevgrad and Study area. Bulgarian metal casting plant others). In the area of enterprise "Radomir Radomir Metals is based the southwestern Metal Industries" soil and water monitoring town of Radomir, Pernik district. The plant at several different points are conducted. has been constructing on 1 800 000 m2 area According to the data of Basin Directorate - and situated at about 50 кm south-west of West Region for wastewater, even those the capital of the Republic of Bulgaria - emitted from the production are in the Sofia, on the road Е79 (Coordinates: emission rate. 42°31'10"N 22°59'12"E). The purification

Table 1. Direct annual pollutant emission standards

Mixed stream wastewater (industrial and Pollutants domestic-faecal) kg.year-1 Total nitrogen 709.40 Total phosphorous 77.45 Arsenic and its compounds 1.54 Cadmium and its compounds 0.15 Chromium and its compounds 1.54 Copper and its compounds 8.52 Mercury and its compounds 0.15 Nickel and its compounds 0.42 Lead and its compounds 1.54 Zinc and its compounds 15.02 Phenols 4.64 Total organic carbon 6954.5 Chlorides - Cyanides 0.57

"Radomir Metal Industries” has owns purified water was the Struma River (II - water treatment plant. Purification is a Second category receiving water). Along the mechanical, biological, as is done drying outlet wastewater to the water treatment and stabilization of sludge. Receiver of point, canal water is intensively used for

Мariana I. Lyubenova, Snejana B. Dineva, Irina B. Karadjova irrigation. Industrial wastewater is water) and with 10 fishes in every aquaria. discharged to the water purification plant The dead fishes have been removed two through a channel running through arable times a day. In 24 h during the test the land. In this regard, research in this work is wastewaters at different dilution have been done to establish whether there is renewed and the alive fishes have been compliance with established standards. moved to the new aquariums. LD50 is Characteristics of the test – objects. The calculated by graphical interpolation (ISO biological tests evaluated the toxicity of 6341/1996, ISO 7346/1:1996). wastewater, polluted air, soil, sediment, etc. The toxicity of Zn (Zn2SO4.7H2O) on P. or a particular pollutant using standard test parva is also been tested using organisms. The latter are exposed to concentrations: 0, 0.001, 0.010, 0.030, 0.040, different concentrations of the substance 0.050 μg.l-1. and report mortality or change in Sample analysis morphology and physiology of organisms. Reagents. All solutions were prepared In determining the toxicity we followed with analytical reagent grade chemicals and standard protocols in order to have ultra-pure water (18 MΩ cm) generated by comparability of results. purifying distilled water with the Milli-QTM For the purpose of study are conducted PLUS system tests with test-objects: Daphnia magna (ISO Nitric acid: Suprapur HNO3 (67% v/v) 6341/1996, 10); Pseudorasbora parva (ISO was purchased from Fluka. 7346/1:1996, 10). The aims of bioassays are The stock standard solutions of Cd, Cu, to determine the substance concentration or Cr, Fe, Hg, Ni, Pb and Zn 1000 mg.l-1 were dilution of water waste, in which occurred Titrisol, Merck in 2% v/v HNO3 and were 50% mortality (LC50) or change in the used to prepare calibration standards. appropriate indicator in the test-organisms Sampling. Fish samples (whole fish for determined time. body) are thoroughly washed with MQ In assessing the toxicity of the effluent water. The fish specimens were dissected of “Radomir Metal Industries” are using and samples of fish gills are quickly change in mortality of D. magna (ISO removed and washed again with MQ water. 6341/1996) and P. parva (ISO 7346/ 1:1996) Fish gills were analyzed as obtained without respectively, at a constant temperature further homogenization. Fish gills (sample 20±2°C. amount between 0.1 and 0.3 g) were By pretesting a series of concentrations digested with nitric acid in MW oven (step 1 (in %) for the final testing are defined: 100, - 250 W for 3 min; step 2 - 400 W for 3 min, 95, 90, 85, 75 и 70 where the D. magna step 3 - 600 W for 3 min), solutions cooled, mortality percentage is rendered in account. transferred in 5 ml volumetric flask and Used for testing fish of species P. parva are diluted up to the mark with Milli-Q water. from one and the same generation and with Instrumentation. Determination of Fe and approximately the same dimensions (length Zn: Flame atomic absorption spectrometric ± 5 mm). The sample has been acclimatized measurements were carried out on a Perkin in aquaria (1 l aerated water for a fish) and Elmer Zeeman 1100 B spectrometer with an normally fed two weeks until the test air/acetylene flame. The instrumental beginning. The fish is not fed and the water parameters were optimized in order to is not aerated during the test. For the final obtain maximum signal-to-noise ratio. testing the following dilutions are used: Determination of Cd and Pb in fish gills: 20%, 40%, 60%, 80%, 100% included smallest Electrothermal atomic absorption lethal and highest not lethal dilution, spectrometric measurements were carried defined by the pre-testing, for 48 h in a two out on a Perkin Elmer Zeeman 3030 repetitions. The tests are conducted in spectrometer with an HGA-600 atomizer. aquariums with an individual volume of 10 Pyrolytic graphite-coated graphite tubes l (with the appropriate dilution of the with integrated platforms were used as effluent and the control with a boiled tap atomizers. Pd as (NH4)2PdCl4 was used as 53

Ecotoxicology Assessment of Waste Water Emitted From Radomir Metal Industries (Bulgaria) modifier for ETAAS measurements of Cd. (e.g. lakes and rivers), making COD a useful Pretreatment temperatures used were 500 measure of water quality. It is expressed in °C for Cd and Pb and atomization milligrams per litre (mg.l-1), which indicates temperatures 1300 °C for Cd and 1900 °C the mass of oxygen consumed per litre of for Pb. solution. Older references may express the Determination of Cd, Cu, Cr, Ni, Pb in units as parts per million (ppm). water samples: Samples measured by Statistics. All obtained results were ETAAS under optimized instrumental statistically processed. It has been shown parameters. that the resulting averages are Determination of Hg in water samples: representative of performance using t-test. Water samples were previously digested The statistical significance level in this study with 1 ml HNO3, Mercury content was was defined at p < 0.05. measured by cold vapour AAS (Varian AA 240 atomic absorption spectrometer Results equipped with a continuous flow VGA-77 Results of samples from wastewater Vapor Generation Accessory) under optimal The table below presents the results of instrumental parameters. some additional analysis of samples of Assessment of water contamination. wastewater and limit concentrations of Biochemical oxygen demand (BOD) and metals under Regulation №6 (Table 2). The chemical oxygen demand (COD) have been marginal limit rate are defined in the determined for the sewage (ISO 6060:1989). particular circumstances of production taken In environmental chemistry, the chemical into account for the manufacture of iron and oxygen demand (COD) test is commonly steel, production of iron and steel castings, used to indirectly measure the amount of cast cars and other non-ferrous metals. organic compounds in water. Most The results show that the resulting applications of COD determine the amount concentrations are within the limits of of organic pollutants found in surface water regulation.

Table 2. Average values with standard deviation of the analyzed parameters of samples effluent compared to the Marginal limit concentrations

Marginal Acidified sample, Not acidified Elements concentrations*, unfiltered, sample, unfiltered, μg.l-1 μg.l-1 μg.l-1 Cd 500 3.2±0.2 2.3±0.2 Cu 500 12.0±2.0 8±1 Cr 500 2.8±0.2 2.1±0.1 Fe 5000 189±14 156±12 Ni 500 4±1 3±1 Pb 200 3.8±0.1 2.1±0.1 Zn 2000 230±15 190±12 Нg 10 <1 <1 pH – 7.92 Chemical oxygen demand (COD) (ВВМ 0208:2001; t = 200±3) - 17.2±1.63 SO42- - 19.5 mg.l-1 Other NH4+ - 0.2 mg.l-1 COD (oximetric) Cl – 10 mg.l-1 indicators NO3- - 5.7 mg.l-1 20.48 mg.l-1 – filtered PO43- - < 0.1 mg.l-1l sample; 73.10 mg.l-1 – unfiltered sample

Мariana I. Lyubenova, Snejana B. Dineva, Irina B. Karadjova

Results from the test with D. magna and P. mately at 86% effluent concentration (un- parva diluted effluent is 100% in the scale, Fig. 2). Daphnia magna Pseudorasbora parva (Temminck & The experiments were carried out with Schlegel, 1846) Stone moroko D. magna, the rate of mortality (LC50) have The dependence of mortality of P. parva been determined. The dose-response curve from effluent with different dilutions is plotted as a cumulative number of dead showed on Fig. 3. organisms by each dose using is represented The dependence of mortality of P. parva (Fig. 1). The dose-effect of the effluent on from different concentration of Zn in the the mortality rate of D. magna correlated water is showed on Fig. 4. well as a linear regression (R2=0.956). Conducting the tests with selected With the increasing of dilution the toxi- dilutions showed that surviving is superior city is reduced. Test results showed that the to that of Daphnia magna, LC50 is reported range of 50% mortality appears approxi- from exposure to raw sewage (100%).

y = 1.6x - 88.182 R2 = 0.956 80

20 Mortality Mortality %

0 0 20 40 60 80 100 120 -10

-20 Dilutions of effluent, %

Fig. 1. Dependence of mortality of D. magna by diluting the effluent

y = 0.5975x + 55.988 2 Log C,% R = 0.956 100

1 -10 0 10 20 30 40 50 60 70 80

Mortality,%

Fig. 2. LC 50 for D. magna under different dilutions of effluent

Ecotoxicology Assessment of Waste Water Emitted From Radomir Metal Industries (Bulgaria)

45 y = 190,48x3 - 285,71x2 + 135,24x + 2E-12 40 R2 = 1 35 30 25 20

Mortality, % Mortality, 15 10 5 0 0,00% 20,00% 40,00% 60,00% 80,00% 100,00% 120,00% Dilutions of effluent, %

Fig. 3. Dependence of mortality of P. parva from effluent with different dilutions

Fig. 4. LC 50 for P. parva under different concentration of Ni in the water (mg.l-1)

Eco-physiological study on the influence of contaminated waters … In all samples of gill Cd is <0.01 ppm, analyses of effluent from the plant of heavy and Pb - <0.1 ppm. For untreated fish, found industry "Radomir Metal Industries", given in the gills content of Fe and Zn is in table 2, showed that the amount of cooper respectively 2.8 and 3.9 ppm. The contents in the waste water is approximately 12±2 of only two of the studied heavy metals in mg.l-1. The acute toxicity has been studied of the gills of P. parva increases - low for Zn heavy metal chromium as well as tannery (1.12 times) and almost double for Fe (1.7 effluent (chromium containing) water has times) compared to untreated specimens. been evaluated by laboratory bioassay experiments for 48 h duration in water flea, Discussion D. magna (TALAPATRA & BANERJEE, 2005). Daphnia magna The median lethal concentration (LC50) D. magna is a Crustacean in the order of value of chromium metal for 48 h duration Cladocera. This aquatic animal extensively was determined to be 0.4027 ppm. Whole used as a test organism in aquatic toxicology effluent toxicity (WET) test confirmed due to their small size, short life cycle and median lethal concentration (LC50) values of amenability to lab culture. D. magna is the tannery effluent water for 48 h duration was most sensitive (PETERS & BERNARDI, 1987). 6.540% dilutions respectively. Chromium D. magna are used as a model organism to content in tannery effluent water was found simulate response of wide ranges of aquatic 3.47 ppm or it is approximately 0.225 ppm invertebrates to toxicants (LANDIS at al., (TALAPATRA & BANERJEE, 2005). The data in 2011; PETERS & BERNARDI, 1987; VEDLEGG, our experiments is showed that the content 2004; VILLAVICENCIO, 2005). The great water of Zn and of Cr in wastewater is relatively flea (D. magna) has been reported as the 0.00189± 0.0014 and 0.0028 ± 0.0002 ppm most sensitive test-object in relation of (table 2). The survival rate of great water different pollutants (organophosphates, flea (D. magna) in our study showed that the organochlorines, heavy metals, pyrethroids effluent from the plant of heavy industry etc.) among all known biological objects "Radomir Metal Industries" is toxic to the including experimental animals (PETERS & test hydro biota, although the concentrations BERNARDI, 1987; Vedlegg for Cyprodinil of individual heavy metals are within the Kilde: Svensk undersøkelse, 2004). The allowed limits, accepted from the Regulation test of mortality has been also reported as № 6. The effluent remains toxic perhaps the the most effective and reliable (HERMENS et combine affect that also occur. It has been al., 1984) with high degree of predictive reported that the joint toxicity of the performance (VILLAVICENCIO, 2005). mixtures of pollutants remains much higher The dose-effect of the effluent on the than that of the individual chemicals mortality rate of D. magna correlated well as (HERMENS et.al., 1984). a linear regression with correlation coefficient R2=0.956 (fig.1). The observed Pseudorasbora parva (Temminck & tendency is a typical pattern for the Schlegel, 1846) Stone moroko dependence of cumulative mortality versus Stone moroko (also known as topmouth environmental concentration or dose of the gudgeon), P. parva, is a fish belonging to the toxicant (LANDIS et al., 2011). The Cyprinid family, native to Asia, but comparable dose-response curve has been introduced and now considered an invasive reported when the effect of different species in Europe (WITKOWSKI, 2006). A concentrations of four compounds on D. review of literature on the ichthyofauna of magna mortality was investigated lowland rivers of Central Europe (KUNGOLOS et al., 2001). (WITKOWSKI et al. 2000, 2004) indicates that Relationship between the concentration the species is now a constant component of of dissolved copper in the overlying water the Central European fauna, and is often a and the mortality of exposed D. magna, dominant. Pseudorasbora parva is a small yielding an estimated LC50 of 26 mg.l-1 planktivore and feeds mainly on aquatic dissolved copper (GILLIS at al., 2006). Water insects, algae, and zooplankton (ZHANG et 57

Ecotoxicology Assessment of Waste Water Emitted From Radomir Metal Industries (Bulgaria) al., 2000; XIE et al., 2000; YANG et al., 2004). In is reported from exposure to raw sewage both its original distribution range and in (100%). Even the concentrations of secondarily invaded areas the stone moroko individual heavy metals are within the inhabits shallow lakes, carp ponds, allowed limits the effluent remains toxic due irrigation canals, ditches, slow sections of to the combine effect of toxicants in waste lowland rivers and their oxbows. It prefers waters that occurs. For the P. parva the much-vegetated places (BERG, 1949; inverse antagonism is observed. Also the KOZLOV, 1974; MUCHACEVA, 1950; gills content of Fe and Zn increases in WITKOWSKI, 2000). treated fishes. The data mortality rate-effluent dilution well correlated with polynomial equation 190,48x3 – 285,71x2 + 135,24x +2E-12, R2=1 (Fig.3). Conducting the tests with selected References dilutions showed that surviving is superior ADRIANO D.C. 2003. Trace Elements in compare to that of daphnia. LC50 is reported Terrestrial Environments: Biogeochemis- from exposure to raw sewage (100%). P. try, Bioavailability and Risks of Metals parva is generally regarded as a pest due to (2nd Edition), Springer, New York. its very high reproductive rate, which gives BANARESCU P.M. 1999. Pseudorasbora bleeker, rise to dense populations of fish 1859. In: The Freshwater Fishes of Europe (WELCOMME, 1988). This species is more Vol 5/I, Cyprinidae 2/I (ed. P.M. resistant than many European fish species to Banarescu). Aula-Verlag GmbH a moderate degree of pollution, elevated Wiebelsheim, pp. 203-224. temperatures, and low water levels. There is BERG L. S. 1949. Ryby presnych vod SSSR i evidence that it also can move a limited sopredelnych stran. 2. Izd. AN SSSR, distance through polluted water Moskva-Leningrad. p. 477- 925. (BANARESCU, 1999). High success in Bulgarian State Standard EN ISO 6341/1996. population growth and the colonization rate Water quality - Determination of the of P. parva could be explained by its wider inhibitory action of toxic substances on ecological physiological tolerance (ROSECCHI the mobility of Daphnia magna Straus et al., 1993; 2001). Our results confirm that (Cladocera, Crustacea) - acute toxicity this species has high tolerance to the water testing. pollution and that wastewater has slight Bulgarian State Standard EN ISO 7346/1: toxic effect on populations of hydro biota. 1996. Water quality - Determination of Nevertheless the tested toxicity of Zn is acute lethal toxicity of substances to expressed by ascending polynomial curve freshwater fish [Brachydanio rerio (fig. 4), where LC50% for 48 h treatment is 33, Hamilton - Buchanan (teleostei; 44 mg.l-1. Perhaps the combine effect Cyprinidae)]. between the toxicants in effluent that occurs GILLIS P.L, WOOD C.M., RANVILLE J.F., is expressed by inverse antagonism CHOW - FRASER P. 2006. Bioavailability (NEWMAN, 2008). of sediment-associated Cu and Zn to Daphnia magna. - Aquatic Toxicology, 77: Conclusions 402–411. The concentrations of the toxicants in HERMENS J., H. CANTON, N. STEYGER, R. the waste waters are under limits. WEGMAN. 1984. Joint effects of a Nevertheless the toxic effect of metal plant mixture of 14 chemicals on mortality sewage on the tested hydro biota has been and inhibition of reproduction of registered. For the great water flea (D. Daphnia magna. - Aquatic Toxicology, magna) 48 h LC50 appears approximately at 5(4): 315-322. 86% effluent concentration. The combined ISO 6060. 1989 toxic effect of the effluent on the two tested [http://www.iso.org/iso/catalogue_d species is different. The surviving of P. parva etail?csnumber=12260] is superior to that of D. magna. The 48 h LC50 58

Мariana I. Lyubenova, Snejana B. Dineva, Irina B. Karadjova

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