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Effects of pulse duration and post-exposure period on the nitrite toxicity to a freshwater amphipod
ARTICLE IN PRESS Ecotoxicology and Environmental Safety 72 (2009) 2005–2008
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of pulse duration and post-exposure period on the nitrite toxicity to a freshwater amphipod Alvaro Alonso a,b,, Julio A. Camargo a a b
Department of Ecology, Faculty of Biology, University of Alcala , 28871 Alcala de Henares, Madrid, Spain Aquatic Ecology and Water Quality Management Group, Wageningen University, P.O. Box 47, 6700 AA Wageningen, The Netherlands
a r t i c l e in fo
abstract
Article history: Received 3 March 2009 Received in revised form 11 June 2009 Accepted 12 June 2009 Available online 28 June 2009
This research assesses the effects of nitrite pulses and post-exposure periods after nitrite exposures on the survival of the freshwater amphipod Eulimnogammarus toletanus. A toxicity bioassay was performed using three different nitrite concentrations (0.5, 5.0 and 10.0 mg/L NO2–N), four pulse exposures (1, 8, 24 and 48 h) for each nitrite concentration, and four post-exposure times until to complete 96 h (i.e., 95, 88, 72 and 48 h, respectively). Our results showed a significant effect of nitrite concentrations, pulses and post-exposure times on the mortality of E. toletanus. The cumulative mortality at the end of pulse and that at the end of post-exposure time (delayed mortality) were different. We conclude that due to the high frequency of intermittent pollution in aquatic ecosystems it is necessary to incorporate the postexposure effects into the traditional toxicological parameters to achieve a more realistic assessment of toxicants, especially at very short-term exposures. & 2009 Elsevier Inc. All rights reserved.
Keywords: Eulimnogammarus toletanus Post-exposure Delayed mortality Intermittent exposure Invertebrate
1. Introduction Aquatic animals are usually exposed to short events of pollution (McCahon and Pascoe, 1990: Ashauer et al., 2007), as a consequence of spray drift, surface runoff, drain flow and accidental spillage from factories or farms. Additionally, when river regulation and pollution sources act together, freshwater organisms can be exposed to daily changes in the concentrations of pollutants as a direct consequence of differential water releases from dams (Camargo, 1996). In all these cases there is a shortterm presence of the toxicant in the water column, being rapidly diluted, photo/bio-degraded or adsorbed by sediments (McCahon and Pascoe, 1990; Handy, 1994; Camargo, 1996). Traditional bioassays focus on the effects of continuous exposure on aquatic animals but ignore the effects after toxicant presence (Abel and Garner, 1986; Ashauer et al., 2007; Newman and Clements, 2008). When the concentration/time of exposure has exceeded the toxicological threshold, deleterious effects may be observed after the toxicant disappearance from water column, due to the permanence of the toxicant within the organism, causing a delayed mortality. On the contrary, when detoxification mechanisms are effective and/or concentration/time of exposure has been low, no effect will be observed after exposure (Mancini, 1983;
Corresponding author at: Department of Ecology, Faculty of Biology, University of Alcala , 28871 Alcala de Henares, Madrid, Spain. Fax: +34 91 885 49 29. Alonso). E-mail address: [email protected] (A.
0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2009.06.008
Pascoe and Shazili, 1986; Ashauer and Brown, 2008). None of these possibilities are tested in traditional toxicological bioassays with aquatic animals (Pascoe and Shazili, 1986). On the other hand, toxic intermittent/pulse studies have been mainly focused in pesticides, metals, acid and ammonia (Pascoe and Shazili, 1986; Alonso and Camargo, 2004; Cold and Forbes, 2004: Ashauer et al., 2006, 2007; Lopez-Mancisidor et al., 2008; Macedo-Sousa et al., 2008). However, there is scarce information on the toxic effects of nitrite (NO 2 ) pulses even though it is a compound with a high toxicity to freshwater animals (Jensen, 2003; Alonso and Camargo, 2006, 2008). This anion is a natural component of the nitrogen cycle, being the intermediate oxidation form between ammonia (NH+4) and nitrate (NO 3 ). Under normal nitrification conditions, nitrite is rapidly oxidized to nitrate by bacteria of the genera Nitrobacter (Philips et al., 2002). However, anthropogenic activities—such as effluents from metal, dyes and celluloid industries, and urban and aquaculture effluents—may alter the nitrification–denitrification process and increase the nitrite concentration, causing toxicity to aquatic animals (Lewis and Morris, 1986; Jensen, 1995; Philips et al., 2002). Nitrite concentration in unpolluted waters ranges from 1 to 3 mg/L, but it can be over 0.9 mg/L NO2–N in polluted surface freshwaters (Von der Wiesche and Wetzel, 1998; Bellos et al., 2004; Fadiran and Mamba, 2005). This rise of nitrite concentrations can be a shortterm event, however, the consequences of post-exposure effects to nitrite have been scarcely studied (Jensen, 1995). This compound can be taken up across gills and accumulated in body fluids, oxidizing the iron of the hemoglobin to methemoglobin, this
ARTICLE IN PRESS 2006
A. Alonso, J.A. Camargo / Ecotoxicology and Environmental Safety 72 (2009) 2005–2008
compromises the oxygen-transport capacity, causing anoxia in fish (Jensen, 1995). In the case of crustacean, NO 2 turns hemocyanin to methemocyanin, although oxygen transport by hemocyanin is less affected by nitrite than that in hemoglobin (Jensen, 1995). Amphipods play a key role in freshwater ecosystems, as they feed on coarse particulate organic matter, allowing the incorporation of this material into the food chain (Cummins, 1979). Additionally, they are an important food source for benthivorous fish (MacNeil et al., 1999). However, there is scarce knowledge on pulse toxicity of nitrite to freshwater amphipods. Therefore, the aim of this study is to assess the effects of short-term nitrite pulses and their respective post-exposure periods on the survival of the freshwater amphipod Eulimnogammarus toletanus (Pinkster & Stock) (Gammaridae, Crustacea). This species was chosen because it has previously showed to be sensitive to the continuous short-term toxicity of nitrite (Alonso and Camargo, 2006).
and 48 h (48 h pulse). Water temperature, pH and dissolved oxygen were monitored daily. Mortality was recorded at the end of each pulse exposure (1, 8 and 24 h) and every 24 h in the case of 48 h pulse. For post-exposure periods, delayed mortality was recorded every 24 h from the beginning of the experiments. At the end of the bioassay, body length of each amphipod, from antennal base to the third uropod, was measured using a stereo microscope equipped with a micrometer. Differences in cumulative mortality between each pulse and control at the end of the bioassay (96 h) for each concentration were compared through a one-way analysis of variance (ANOVA, Dunnett test). This comparison identifies concentrations and pulses where mortality is significantly higher than in the control. After that, the comparison to assess the effects of concentration, pulse duration and post-exposure time on the cumulative mortality at the end of pulse and in the delayed mortality after 96 h was conducted through a three-way ANOVA. Differences in mean body length between all treatments (including the control), and differences in actual nitrite concentrations (between treatments with the same nominal concentrations) were compared with a one-way analysis of variance (ANOVA; Tukey test). Data were tested for heterogeneity of variance using Levene’s test. Significance was accepted at Po0.05. All statistical analyses were performed using SPSS 12.0 software.
3. Results
Amphipods were collected from a natural upper reach of the Henares River (Central Spain). Once in the laboratory, animals were progressively acclimatized to test water (bottled drinking water without chlorine) for 7 days before starting the experiment. The physical–chemical properties of test water were: 369.879.6 mS/ cm for conductivity, 29.070.8 mg/L of chloride (Cl), 8.070.2 for pH, 6.770.5 mg/ L O2 for dissolved oxygen, and 15.371.1 1C for water temperature. Amphipods were fed during acclimatization with stream-conditioned poplar (Populus sp.). Precopulatory pairs were not used in the bioassay. A nitrite toxicity bioassay was conducted in triplicate using glass vessels (0.1 L). Eight randomly selected amphipods were situated in each vessel. Experimental design is presented in Table 1. Three nominal concentrations of nitrite were used: 0.5, 5.0 and 10.0 mg/L NO2–N. These concentrations were selected on the basis of previous studies (Alonso and Camargo 2006, 2008) that showed lethal effects for the highest concentrations at continuous short-term exposures. All nominal concentrations were prepared from a stock solution of 100 mg/L of NO2–N. This stock was prepared daily by dissolving the required amount of sodium nitrite (NaNO2) (SIGMA, Germany, Lot no. 97H1563, reported purity of 99.5%, previously dried at 60 1C) in 1000 mL of test water. For each concentration four exposure times (i.e. pulses) were selected: 1, 8, 24 and 48 h. After the end of each pulse and for each concentration, amphipods were immediately transferred to control water for post-exposure periods of 95, 88, 72 and 48 h, respectively. Therefore, the total time of exposure and post-exposure was 96 h for each concentration/pulse. Control animals were treated in the same way after 24 h (from test water to new test water). Test solutions, including the control and post-exposure periods, were renewed daily. Actual concentrations of nitrite were measured by spectrophotometry (detection limit ¼ 0.005 mg/L NO2–N) (American Public Health Association, 1995) after 0 and 1 h (1 h pulse), 0 and 8 h (8 h pulse), 0 and 24 h (24 h pulse), and 0, 24 (before and after solution renewal)
Mean mortality in control vessels was less than 10%. Significant differences were neither found between treatments (including control) for the mean body length of amphipods nor between mean actual nitrite concentrations between treatments with the same nominal nitrite concentration (P40.05; Tukey test) (Table 1). As nominal concentrations were very similar to actual ones (Table 1) all concentrations cited in the results are nominal concentrations. Cumulative mortality was a function of nitrite concentration and pulse duration after 96 h. Nitrite concentration of 0.5 mg/L 120 % cumulative mortality
2. Materials and methods
100
0.5 mg/L NO2-N 5.0 mg/L NO2-N 10.0 mg/L NO2-N
* *
80
*
60
* *
40 20 0
Control
1h
8h
24h
48h
Pulse Treatment Table 1 Pulse design used to evaluate the effect of nitrite on Eulimnogammarus toletanus. Pulse
Nitrite (mg/L NO2–N)
Exposure time (h)
Post-exposure (h)
Body length (mm)
Control 1h 8h 24 h 48 h 1h 8h 24 h 48 h 1h 8h 24 h 48 h
o0.005 0.5070.01 0.5170.01 0.4970.01 0.5270.01 5.0370.05 5.0570.06 5.0070.08 5.1570.06 10.070.16 10.270.10 10.170.12 10.270.16
– 1 8 24 48 1 8 24 48 1 8 24 48
– 95 88 72 48 95 88 72 48 95 88 72 48
6.070.8 5.770.7 5.570.8 6.070.6 5.770.6 5.770.7 5.770.5 5.770.7 5.970.6 5.770.8 5.670.7 6.170.8 5.970.5
Mean body length (mm) and actual nitrite concentration (mg/L NO2–N) (7SD) are showed for each pulse. No significant differences were found between treatments for body length or nitrite concentration between treatment with the same nominal concentration (P40.05; Tukey test).
Fig. 1. Mean+standard deviation for cumulative mortalities of amphipods after the end of the test (exposure+post-exposure ¼ 96 h) for each pulse and concentration. Asterisks show significant difference in mortality between each pulse treatment and control (ANOVA; Dunnett test; Po0.05).
Table 2 Summary of the three-way analysis of variance of the effect of post-exposure, pulse (8, 24 and 48 h), nitrite concentration (5.0 and 10.0 mg/L NO2–N) and their interactions on the end-of-test mortality of the freshwater amphipod Eulimnogammarus toletanus. Source of variation
F
P
Post-exposure Pulse (8, 24 and 48 h) Concentration (5.0 and 10.0 mg/L NO2–N) Pulse Concentration Post-exposure Pulse Post-exposure Concentration Post-exposure Pulse Concentration
18.6 53.8 74.3 10.6 2.03 0.129 1.20
0.000 0.000 0.000 0.001 0.153 0.723 0.321
Bold letters show the P values that are significant. All P values are less or equal to 0.001.
ARTICLE IN PRESS A. Alonso, J.A. Camargo / Ecotoxicology and Environmental Safety 72 (2009) 2005–2008
120
% Mortality
100 80
5.0 mg/L NO2-N
2007
120
10.0 mg/L NO2-N
100
At end of exposure At end of 96 hr test
80
60
60
40
40
20
20 0
0 Control
8h
24h
48h
Pulse
Control
8h
24h
48h
Pulse
Fig. 2. Mean cumulative mortalities of amphipods (7SD) after the end of pulse exposure (8, 24 and 48 h, black bars) and post-exposure time (88, 72 and 48 h, white bars) for each nitrite concentration ((A) 5.0 and (B) 10.0 mg/L NO2–N).
NO2–N did not cause a significant increase in cumulative mortality with respect to the control for any pulse (P40.05; Dunnett test) (Fig. 1). In the case of 5.0 mg/L NO2–N, mortality was significantly higher than that in the control only for 24 and 48 h pulses after 96 h (Po0.05; Dunnett test). Concentration of 10.0 mg/L NO2–N caused a significant increase in mortality for 8, 24 and 48 h pulses after 96 h (Po0.05; Dunnett test). As a consequence of these results, nitrite concentrations of 5.0 and 10.0 mg/L NO2–N, and pulse durations of 8, 24 and 48 h were selected for the three-way analysis of variance ( ¼ treatments with significant effects) (Table 2). Post-exposure caused a significant effect in mortality (Po0.001; ANOVA) because, after the end of post-exposure time (96 h) delayed mortality was higher than mortality after pulse exposure (Fig. 2). Significant effects were also found to both pulse duration and concentration (Po0.001; ANOVA) (Table 2, Fig. 2). The interaction of pulse duration and nitrite concentration was also significant, since for the different pulses the trend in mortality was different between nitrite concentrations (Fig. 2). The other interactions were not significant (P40.05; ANOVA) (Table 2).
4. Discussion Several factors are important in pulse toxicological studies, such as toxic concentration, pulse duration, recovery time and frequency of pulses (Handy, 1994; Camargo, 1996; Naddy and Klaine, 2001). In our study, the tested factors (post-exposure, pulse duration and concentration) had effects on the short-term toxicity of nitrite to E. toletanus. The cumulative mortality was lower at the end of pulse than that at the end of post-exposure time. There are two main likely causes for that. The first cause can be that a part of the tested amphipods could not metabolize or excrete the toxicant during the post-exposure period, causing hypoxia on the tissues of aquatic animals (Jensen, 2003). These hypoxia events can damage tissues, which can take considerable time to repair, increasing the mortality after toxicant exposure. A similar mortality increasing in the post-exposure time has been observed for other common toxicants that caused pervasive changes or damage on tissues (e.g. copper, sodium chloride, etc.) (Newman and Clements, 2008). A second cause may be that high nitrite concentrations reduce the functionality of hemocyanin, causing mortality by anoxia in the most sensitive amphipods during the post-exposure period. Obviously, both causes (damage on tissues and low availability of oxygen) are connected, and can happen together through time. Additionally, nitrite toxicity to aquatic animals can be attributable to other physiological mechanisms, such us alterations of electrolyte or acid–base balance (Jensen, 1995, 2003), than can also contribute to the high adverse effect during the post-exposure periods.
A lost of nitrite across gills and/or in the urine have been cited as physiological protective mechanisms in aquatic animals (Jensen, 1995, 2003). These mechanisms could be the reason for the survival of a part of the exposed amphipods, both during the exposure and post-exposure periods. Additionally, the methemoglobin reductasa can recover the level of hemoglobin in fish exposed to nitrite (Knudsen and Jensen, 1997). A similar enzymatic recovery mechanism to hemocyanin can be active in amphipods. However, there is a lack of knowledge of nitrite effects on the physiology of freshwater amphipods and the effects of post-exposure periods in the detoxification process after nitrite exposure in this animal group. Understanding the mechanistic causes of the observed effects in amphipods after nitrite exposure requires further studies. On the other hand, in standard toxicity tests animals are exposed to a constant toxic concentration until toxicological effects are observed (mortality, immobility, alteration of feeding activity, etc.) (McCahon and Pascoe, 1990; Cold and Forbes, 2004). As a consequence the toxicological calculated parameters—such as median lethal time or lethal concentration—do not consider the delayed effects, especially when only short-exposure bioassays are conducted (e.g. 48 h or less). Therefore, the environmental quality standards and permissible safe levels for aquatic ecosystems are established from results of traditional tests (LC50, EC50, LOEC and NOEC values) (Pascoe and Shazili, 1986; Fisher et al., 2003), even though natural ecosystems show seasonal and daily changes in nitrite concentrations (Von der Wiesche and Wetzel, 1998; Scholefield et al., 2005). Toxicological parameters from pulse exposure tests should be incorporated into the ecological risk assessment or used as environmental quality standards for toxicants with a short presence on the water column (e.g., pesticides, nitrite, etc.). Therefore, further research is necessary to know how to include the delayed effects in traditional toxicological parameters (LC50, EC50 or LOEC). One possibility is to include the post-exposure effects (e.g. delayed mortality) in the calculation of the toxicological parameters (e.g. LC50, EC50 or LOEC), given a more realistic assessment of the effects of toxicants. The inclusion of delayed effects may produce a reduction in the permissible or safe concentrations for aquatic environments, especially in toxicants with a rapid deleterious acute effect on animal physiology (as nitrite). In this way, traditional toxicological parameters might be related to exposure time effects (e.g. instantaneous LC50 or LOEC) and post-exposure time effects (e.g. delayed LC50 or LOEC for a given time). A similar toxicological parameter (as median post-exposure lethal time, peLT50) was proposed by Pascoe and Shazili (1986) to assess lethal time of cadmium to rainbow trout fry. Alternatively, bioassays can be conducted with a sufficient duration to observe both exposure and post-exposure effects (e.g. several hours or days after the end of exposure). Additionally, several toxicokinetic and toxicodynamic models have been proposed as a tool for mechanism-based
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ecotoxicology, allowing the simulation of the dynamic of toxicants in the whole organisms or to predict effects (Hickie et al., 1995; Ashauer et al., 2006, 2007; Unger et al., 2007; Ashauer and Brown 2008). As organism concentration is surrogated to the environmental concentration, these models can contribute to describe the dynamics of the deleterious effects and delayed mortality. Therefore, they can be a useful tool to assess pulse and delayed effects when only continuous exposure data are available.
5. Conclusions We conclude that the pulse duration and post-exposure were two factors that influenced in the lethal response of E. toletanus to nitrite, being the mortality at the end of the nitrite exposure lower than that after the end of the post-exposure period. Additionally, due to the high frequency of intermittent pollution in aquatic ecosystems, it would be necessary to incorporate the delayed mortality and other possible delayed effects into the traditional toxicological parameters and ecological risk assessment. This is especially relevant for very short-life toxicants (e.g. quickly oxidized, photo-degraded, etc.), that can stay in the water column for a few hours/days.
Acknowledgments This research was financed by the Spanish Ministry of Science and Technology (Research Project REN2001-1008) and Alcala University. Dr. A. Alonso was supported by the Council of CastillaLa Mancha Community and Alcala University (predoctoral grants). He is currently supported by the Spanish Ministry of Science and Innovation (Juan de la Cierva contract). Additionally, he has received a grant from the Wageningen Institute for Environment and Climate Research (WIMEK) to stay at the Aquatic Ecology and Water Quality Management Group (Wageningen University, The Netherlands). Our sincere gratitude to Pilar for correcting the English text and to Marcos de la Puente for his help with the taxonomic identification. Furthermore, we are grateful for two anonymous reviewers for their valuable comments to improve this manuscript. References Abel, P.D., Garner, S.M., 1986. Comparisons of median survival times and median lethal exposure times for Gammarus pulex exposed to cadmium, permethrin and cyanide. Water Res. 20, 579–582. Alonso, A., Camargo, J.A., 2004. Sub-lethal responses of the aquatic snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca) to unionized ammonia: a tolerant invading species. Fresen. Environ. Bull. 13, 607–615. Alonso, A., Camargo, J.A., 2006. Toxicity of nitrite to three species of freshwater invertebrates. Environ. Toxicol. 21, 90–94. Alonso, A., Camargo, J.A., 2008. Ameliorating effect of chloride on nitrite toxicity to freshwater invertebrates with different physiology: a comparative study between amphipods and planarians. Arch. Environ. Contam. Toxicol. 54, 259–265. American Public Health Association, 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. American Public Association, Washington, DC, USA.
Ashauer, R., Brown, C., 2008. Toxicodynamic assumptions in ecotoxicological hazard models. Environ. Toxicol. Chem. 27, 1817–1821. Ashauer, R., Bosall, A.B.A., Brown, C., 2006. Predicting effects on aquatic organisms from fluctuating or pulsed exposure to pesticides. Environ. Toxicol. Chem. 25, 1899–1912. Ashauer, R., Bosall, A.B.A., Brown, C., 2007. Simulating toxicity of carbaryl to Gammarus pulex after sequential pulsed exposure. Environ. Sci. Technol. 41, 5528–5534. Bellos, D., Sawidis, T., Tsekos, I., 2004. Nutrient chemistry of river Pinios (Thessalia, Greece). Environ. Int. 30, 105–115. Camargo, J.A., 1996. Comparing levels of pollutants in regulated rivers with safe concentrations of pollutants for fishes: a case study. Chemosphere 33, 81–90. Cold, A., Forbes, V.E., 2004. Consequences of a short pulse of pesticide exposure for survival and reproduction of Gammarus pulex. Aquat. Toxicol. 67, 287–299. Cummins, K.W., 1979. Feeding ecology of stream invertebrates. Ann. Rev. Ecol. Syst. 10, 147–172. Fadiran, A.O., Mamba, S.M., 2005. Analysis of nitrates and nitrites in some water and factory effluent samples from some cities in Swaziland. Bull. Chem. Soc. Ethiopia 19, 35–44. Fisher, D.J., Burton, D.T., Yonkos, L.T., Turley, S.D., Ziegler, G.P., Turley, B.S., 2003. Derivation of acute ecological risk criteria for chlorite in freshwater ecosystems. Water Res. 37, 4359–4368. Handy, R.D., 1994. Intermittent exposure to aquatic pollutants: assessment, toxicity and sublethal responses in fish and invertebrates: mini review. Comp. Biochem. Physiol. 107C, 171–184. Hickie, B.E., McCarty, L.S., Dixon, G.D., 1995. A residue-based toxicokinetic model for pulse-exposure toxicity in aquatic systems. Environ. Toxicol. Chem. 14, 2187–2197. Jensen, F.B., 1995. Uptake and effects of nitrite and nitrate in animals. In: Walsh, P.J., Wright, P. (Eds.), Nitrogen Metabolism and Excretion. CRC Press, Boca Raton, pp. 289–303. Jensen, F.B., 2003. Nitrite disrupts multiple physiological functions in aquatic animals. Comp. Biochem. Physiol. 135A, 9–24. Knudsen, P.K., Jensen, F.B., 1997. Recovery from nitrite induced methaemoglobinaemia and potassium balance disturbances in carp. Fish. Physiol. Biochem. 16, 1–10. Lewis, W.M., Morris, D.P., 1986. Toxicity of nitrite to fish: a review. Trans. Am. Fish. Soc. 115, 183–195. Lopez-Mancisidor, P., Carbonell, G., Fernandez, C., Tarazona, J.V., 2008. Ecological impact of repeated applications of chlorpyrifos on zooplankton community in mesocosms under Mediterranean conditions. Ecotoxicology 17, 811–825. Macedo-Sousa, J.A., Gerhardt, A., Brett, C.M.A., Nogueira, A.J.A., Soares, A.M.V.M., 2008. Behavioural responses of indigenous benthic invertebrates (Echinogammarus meridionalis, Hydropsyche pellucidula and Choroterpes picteti) to a pulse of acid mine drainage: a laboratorial study. Environ. Pollut. 156, 966–973. MacNeil, C., Dick, J.T.A., Elwood, R.W., 1999. The dynamics of predation on Gammarus spp. (Crustacea: Amphipoda). Biol. Rev. 74, 375–395. Mancini, J.L., 1983. A method for calculating effects, on aquatic organisms, of time varying concentrations. Water Res. 17, 1355–1362. McCahon, C.P., Pascoe, D., 1990. Episodic pollution: causes, toxicological effects and ecological significance. Funct. Ecol. 4, 375–383. Naddy, R.B., Klaine, S.J., 2001. Effect of pulse frequency and interval on the toxicity of chlorpyrifos to Daphnia magna. Chemosphere 45, 497–506. Newman, M.C., Clements, W.H., 2008. Ecotoxicology: A Comprehensive Treatment. CRC Press, New York. Pascoe, D., Shazili, N.A.M., 1986. Episodic pollution—a comparison of brief and continuous exposure of rainbow trout to cadmium. Ecotoxicol. Environ. Saf. 12, 189–198. Philips, S., Laanbroek, H.J., Verstraete, W., 2002. Origin, causes and effects of increased nitrite concentrations in aquatic environments. Rev. Environ. Sci. Biotechnol. 1, 115–141. Scholefield, D., Le Goff, T., Braven, J., Ebdon, L., Long, T., Butler, M., 2005. Concerted diurnal patterns in riverine nutrient concentrations and physical conditions. Sci. Total Environ. 344, 201–210. Unger, M.A., Newman, M.C., Vadas, G.G., 2007. Predicting survival of grass shrimp (Palaemonetes pugio) during ethylnaphthalene, dimethylnaphthalene, and phenanthrene exposures differing in concentration and duration. Environ. Toxicol. Chem. 26, 528–534. Von der Wiesche, M., Wetzel, A., 1998. Temporal and spatial dynamics of nitrite accumulation in the river Lahn. Water Res. 32, 1653–1661.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of pulse duration and post-exposure period on the nitrite toxicity to a freshwater amphipod Alvaro Alonso a,b,, Julio A. Camargo a a b
Department of Ecology, Faculty of Biology, University of Alcala , 28871 Alcala de Henares, Madrid, Spain Aquatic Ecology and Water Quality Management Group, Wageningen University, P.O. Box 47, 6700 AA Wageningen, The Netherlands
a r t i c l e in fo
abstract
Article history: Received 3 March 2009 Received in revised form 11 June 2009 Accepted 12 June 2009 Available online 28 June 2009
This research assesses the effects of nitrite pulses and post-exposure periods after nitrite exposures on the survival of the freshwater amphipod Eulimnogammarus toletanus. A toxicity bioassay was performed using three different nitrite concentrations (0.5, 5.0 and 10.0 mg/L NO2–N), four pulse exposures (1, 8, 24 and 48 h) for each nitrite concentration, and four post-exposure times until to complete 96 h (i.e., 95, 88, 72 and 48 h, respectively). Our results showed a significant effect of nitrite concentrations, pulses and post-exposure times on the mortality of E. toletanus. The cumulative mortality at the end of pulse and that at the end of post-exposure time (delayed mortality) were different. We conclude that due to the high frequency of intermittent pollution in aquatic ecosystems it is necessary to incorporate the postexposure effects into the traditional toxicological parameters to achieve a more realistic assessment of toxicants, especially at very short-term exposures. & 2009 Elsevier Inc. All rights reserved.
Keywords: Eulimnogammarus toletanus Post-exposure Delayed mortality Intermittent exposure Invertebrate
1. Introduction Aquatic animals are usually exposed to short events of pollution (McCahon and Pascoe, 1990: Ashauer et al., 2007), as a consequence of spray drift, surface runoff, drain flow and accidental spillage from factories or farms. Additionally, when river regulation and pollution sources act together, freshwater organisms can be exposed to daily changes in the concentrations of pollutants as a direct consequence of differential water releases from dams (Camargo, 1996). In all these cases there is a shortterm presence of the toxicant in the water column, being rapidly diluted, photo/bio-degraded or adsorbed by sediments (McCahon and Pascoe, 1990; Handy, 1994; Camargo, 1996). Traditional bioassays focus on the effects of continuous exposure on aquatic animals but ignore the effects after toxicant presence (Abel and Garner, 1986; Ashauer et al., 2007; Newman and Clements, 2008). When the concentration/time of exposure has exceeded the toxicological threshold, deleterious effects may be observed after the toxicant disappearance from water column, due to the permanence of the toxicant within the organism, causing a delayed mortality. On the contrary, when detoxification mechanisms are effective and/or concentration/time of exposure has been low, no effect will be observed after exposure (Mancini, 1983;
Corresponding author at: Department of Ecology, Faculty of Biology, University of Alcala , 28871 Alcala de Henares, Madrid, Spain. Fax: +34 91 885 49 29. Alonso). E-mail address: [email protected] (A.
0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2009.06.008
Pascoe and Shazili, 1986; Ashauer and Brown, 2008). None of these possibilities are tested in traditional toxicological bioassays with aquatic animals (Pascoe and Shazili, 1986). On the other hand, toxic intermittent/pulse studies have been mainly focused in pesticides, metals, acid and ammonia (Pascoe and Shazili, 1986; Alonso and Camargo, 2004; Cold and Forbes, 2004: Ashauer et al., 2006, 2007; Lopez-Mancisidor et al., 2008; Macedo-Sousa et al., 2008). However, there is scarce information on the toxic effects of nitrite (NO 2 ) pulses even though it is a compound with a high toxicity to freshwater animals (Jensen, 2003; Alonso and Camargo, 2006, 2008). This anion is a natural component of the nitrogen cycle, being the intermediate oxidation form between ammonia (NH+4) and nitrate (NO 3 ). Under normal nitrification conditions, nitrite is rapidly oxidized to nitrate by bacteria of the genera Nitrobacter (Philips et al., 2002). However, anthropogenic activities—such as effluents from metal, dyes and celluloid industries, and urban and aquaculture effluents—may alter the nitrification–denitrification process and increase the nitrite concentration, causing toxicity to aquatic animals (Lewis and Morris, 1986; Jensen, 1995; Philips et al., 2002). Nitrite concentration in unpolluted waters ranges from 1 to 3 mg/L, but it can be over 0.9 mg/L NO2–N in polluted surface freshwaters (Von der Wiesche and Wetzel, 1998; Bellos et al., 2004; Fadiran and Mamba, 2005). This rise of nitrite concentrations can be a shortterm event, however, the consequences of post-exposure effects to nitrite have been scarcely studied (Jensen, 1995). This compound can be taken up across gills and accumulated in body fluids, oxidizing the iron of the hemoglobin to methemoglobin, this
ARTICLE IN PRESS 2006
A. Alonso, J.A. Camargo / Ecotoxicology and Environmental Safety 72 (2009) 2005–2008
compromises the oxygen-transport capacity, causing anoxia in fish (Jensen, 1995). In the case of crustacean, NO 2 turns hemocyanin to methemocyanin, although oxygen transport by hemocyanin is less affected by nitrite than that in hemoglobin (Jensen, 1995). Amphipods play a key role in freshwater ecosystems, as they feed on coarse particulate organic matter, allowing the incorporation of this material into the food chain (Cummins, 1979). Additionally, they are an important food source for benthivorous fish (MacNeil et al., 1999). However, there is scarce knowledge on pulse toxicity of nitrite to freshwater amphipods. Therefore, the aim of this study is to assess the effects of short-term nitrite pulses and their respective post-exposure periods on the survival of the freshwater amphipod Eulimnogammarus toletanus (Pinkster & Stock) (Gammaridae, Crustacea). This species was chosen because it has previously showed to be sensitive to the continuous short-term toxicity of nitrite (Alonso and Camargo, 2006).
and 48 h (48 h pulse). Water temperature, pH and dissolved oxygen were monitored daily. Mortality was recorded at the end of each pulse exposure (1, 8 and 24 h) and every 24 h in the case of 48 h pulse. For post-exposure periods, delayed mortality was recorded every 24 h from the beginning of the experiments. At the end of the bioassay, body length of each amphipod, from antennal base to the third uropod, was measured using a stereo microscope equipped with a micrometer. Differences in cumulative mortality between each pulse and control at the end of the bioassay (96 h) for each concentration were compared through a one-way analysis of variance (ANOVA, Dunnett test). This comparison identifies concentrations and pulses where mortality is significantly higher than in the control. After that, the comparison to assess the effects of concentration, pulse duration and post-exposure time on the cumulative mortality at the end of pulse and in the delayed mortality after 96 h was conducted through a three-way ANOVA. Differences in mean body length between all treatments (including the control), and differences in actual nitrite concentrations (between treatments with the same nominal concentrations) were compared with a one-way analysis of variance (ANOVA; Tukey test). Data were tested for heterogeneity of variance using Levene’s test. Significance was accepted at Po0.05. All statistical analyses were performed using SPSS 12.0 software.
3. Results
Amphipods were collected from a natural upper reach of the Henares River (Central Spain). Once in the laboratory, animals were progressively acclimatized to test water (bottled drinking water without chlorine) for 7 days before starting the experiment. The physical–chemical properties of test water were: 369.879.6 mS/ cm for conductivity, 29.070.8 mg/L of chloride (Cl), 8.070.2 for pH, 6.770.5 mg/ L O2 for dissolved oxygen, and 15.371.1 1C for water temperature. Amphipods were fed during acclimatization with stream-conditioned poplar (Populus sp.). Precopulatory pairs were not used in the bioassay. A nitrite toxicity bioassay was conducted in triplicate using glass vessels (0.1 L). Eight randomly selected amphipods were situated in each vessel. Experimental design is presented in Table 1. Three nominal concentrations of nitrite were used: 0.5, 5.0 and 10.0 mg/L NO2–N. These concentrations were selected on the basis of previous studies (Alonso and Camargo 2006, 2008) that showed lethal effects for the highest concentrations at continuous short-term exposures. All nominal concentrations were prepared from a stock solution of 100 mg/L of NO2–N. This stock was prepared daily by dissolving the required amount of sodium nitrite (NaNO2) (SIGMA, Germany, Lot no. 97H1563, reported purity of 99.5%, previously dried at 60 1C) in 1000 mL of test water. For each concentration four exposure times (i.e. pulses) were selected: 1, 8, 24 and 48 h. After the end of each pulse and for each concentration, amphipods were immediately transferred to control water for post-exposure periods of 95, 88, 72 and 48 h, respectively. Therefore, the total time of exposure and post-exposure was 96 h for each concentration/pulse. Control animals were treated in the same way after 24 h (from test water to new test water). Test solutions, including the control and post-exposure periods, were renewed daily. Actual concentrations of nitrite were measured by spectrophotometry (detection limit ¼ 0.005 mg/L NO2–N) (American Public Health Association, 1995) after 0 and 1 h (1 h pulse), 0 and 8 h (8 h pulse), 0 and 24 h (24 h pulse), and 0, 24 (before and after solution renewal)
Mean mortality in control vessels was less than 10%. Significant differences were neither found between treatments (including control) for the mean body length of amphipods nor between mean actual nitrite concentrations between treatments with the same nominal nitrite concentration (P40.05; Tukey test) (Table 1). As nominal concentrations were very similar to actual ones (Table 1) all concentrations cited in the results are nominal concentrations. Cumulative mortality was a function of nitrite concentration and pulse duration after 96 h. Nitrite concentration of 0.5 mg/L 120 % cumulative mortality
2. Materials and methods
100
0.5 mg/L NO2-N 5.0 mg/L NO2-N 10.0 mg/L NO2-N
* *
80
*
60
* *
40 20 0
Control
1h
8h
24h
48h
Pulse Treatment Table 1 Pulse design used to evaluate the effect of nitrite on Eulimnogammarus toletanus. Pulse
Nitrite (mg/L NO2–N)
Exposure time (h)
Post-exposure (h)
Body length (mm)
Control 1h 8h 24 h 48 h 1h 8h 24 h 48 h 1h 8h 24 h 48 h
o0.005 0.5070.01 0.5170.01 0.4970.01 0.5270.01 5.0370.05 5.0570.06 5.0070.08 5.1570.06 10.070.16 10.270.10 10.170.12 10.270.16
– 1 8 24 48 1 8 24 48 1 8 24 48
– 95 88 72 48 95 88 72 48 95 88 72 48
6.070.8 5.770.7 5.570.8 6.070.6 5.770.6 5.770.7 5.770.5 5.770.7 5.970.6 5.770.8 5.670.7 6.170.8 5.970.5
Mean body length (mm) and actual nitrite concentration (mg/L NO2–N) (7SD) are showed for each pulse. No significant differences were found between treatments for body length or nitrite concentration between treatment with the same nominal concentration (P40.05; Tukey test).
Fig. 1. Mean+standard deviation for cumulative mortalities of amphipods after the end of the test (exposure+post-exposure ¼ 96 h) for each pulse and concentration. Asterisks show significant difference in mortality between each pulse treatment and control (ANOVA; Dunnett test; Po0.05).
Table 2 Summary of the three-way analysis of variance of the effect of post-exposure, pulse (8, 24 and 48 h), nitrite concentration (5.0 and 10.0 mg/L NO2–N) and their interactions on the end-of-test mortality of the freshwater amphipod Eulimnogammarus toletanus. Source of variation
F
P
Post-exposure Pulse (8, 24 and 48 h) Concentration (5.0 and 10.0 mg/L NO2–N) Pulse Concentration Post-exposure Pulse Post-exposure Concentration Post-exposure Pulse Concentration
18.6 53.8 74.3 10.6 2.03 0.129 1.20
0.000 0.000 0.000 0.001 0.153 0.723 0.321
Bold letters show the P values that are significant. All P values are less or equal to 0.001.
ARTICLE IN PRESS A. Alonso, J.A. Camargo / Ecotoxicology and Environmental Safety 72 (2009) 2005–2008
120
% Mortality
100 80
5.0 mg/L NO2-N
2007
120
10.0 mg/L NO2-N
100
At end of exposure At end of 96 hr test
80
60
60
40
40
20
20 0
0 Control
8h
24h
48h
Pulse
Control
8h
24h
48h
Pulse
Fig. 2. Mean cumulative mortalities of amphipods (7SD) after the end of pulse exposure (8, 24 and 48 h, black bars) and post-exposure time (88, 72 and 48 h, white bars) for each nitrite concentration ((A) 5.0 and (B) 10.0 mg/L NO2–N).
NO2–N did not cause a significant increase in cumulative mortality with respect to the control for any pulse (P40.05; Dunnett test) (Fig. 1). In the case of 5.0 mg/L NO2–N, mortality was significantly higher than that in the control only for 24 and 48 h pulses after 96 h (Po0.05; Dunnett test). Concentration of 10.0 mg/L NO2–N caused a significant increase in mortality for 8, 24 and 48 h pulses after 96 h (Po0.05; Dunnett test). As a consequence of these results, nitrite concentrations of 5.0 and 10.0 mg/L NO2–N, and pulse durations of 8, 24 and 48 h were selected for the three-way analysis of variance ( ¼ treatments with significant effects) (Table 2). Post-exposure caused a significant effect in mortality (Po0.001; ANOVA) because, after the end of post-exposure time (96 h) delayed mortality was higher than mortality after pulse exposure (Fig. 2). Significant effects were also found to both pulse duration and concentration (Po0.001; ANOVA) (Table 2, Fig. 2). The interaction of pulse duration and nitrite concentration was also significant, since for the different pulses the trend in mortality was different between nitrite concentrations (Fig. 2). The other interactions were not significant (P40.05; ANOVA) (Table 2).
4. Discussion Several factors are important in pulse toxicological studies, such as toxic concentration, pulse duration, recovery time and frequency of pulses (Handy, 1994; Camargo, 1996; Naddy and Klaine, 2001). In our study, the tested factors (post-exposure, pulse duration and concentration) had effects on the short-term toxicity of nitrite to E. toletanus. The cumulative mortality was lower at the end of pulse than that at the end of post-exposure time. There are two main likely causes for that. The first cause can be that a part of the tested amphipods could not metabolize or excrete the toxicant during the post-exposure period, causing hypoxia on the tissues of aquatic animals (Jensen, 2003). These hypoxia events can damage tissues, which can take considerable time to repair, increasing the mortality after toxicant exposure. A similar mortality increasing in the post-exposure time has been observed for other common toxicants that caused pervasive changes or damage on tissues (e.g. copper, sodium chloride, etc.) (Newman and Clements, 2008). A second cause may be that high nitrite concentrations reduce the functionality of hemocyanin, causing mortality by anoxia in the most sensitive amphipods during the post-exposure period. Obviously, both causes (damage on tissues and low availability of oxygen) are connected, and can happen together through time. Additionally, nitrite toxicity to aquatic animals can be attributable to other physiological mechanisms, such us alterations of electrolyte or acid–base balance (Jensen, 1995, 2003), than can also contribute to the high adverse effect during the post-exposure periods.
A lost of nitrite across gills and/or in the urine have been cited as physiological protective mechanisms in aquatic animals (Jensen, 1995, 2003). These mechanisms could be the reason for the survival of a part of the exposed amphipods, both during the exposure and post-exposure periods. Additionally, the methemoglobin reductasa can recover the level of hemoglobin in fish exposed to nitrite (Knudsen and Jensen, 1997). A similar enzymatic recovery mechanism to hemocyanin can be active in amphipods. However, there is a lack of knowledge of nitrite effects on the physiology of freshwater amphipods and the effects of post-exposure periods in the detoxification process after nitrite exposure in this animal group. Understanding the mechanistic causes of the observed effects in amphipods after nitrite exposure requires further studies. On the other hand, in standard toxicity tests animals are exposed to a constant toxic concentration until toxicological effects are observed (mortality, immobility, alteration of feeding activity, etc.) (McCahon and Pascoe, 1990; Cold and Forbes, 2004). As a consequence the toxicological calculated parameters—such as median lethal time or lethal concentration—do not consider the delayed effects, especially when only short-exposure bioassays are conducted (e.g. 48 h or less). Therefore, the environmental quality standards and permissible safe levels for aquatic ecosystems are established from results of traditional tests (LC50, EC50, LOEC and NOEC values) (Pascoe and Shazili, 1986; Fisher et al., 2003), even though natural ecosystems show seasonal and daily changes in nitrite concentrations (Von der Wiesche and Wetzel, 1998; Scholefield et al., 2005). Toxicological parameters from pulse exposure tests should be incorporated into the ecological risk assessment or used as environmental quality standards for toxicants with a short presence on the water column (e.g., pesticides, nitrite, etc.). Therefore, further research is necessary to know how to include the delayed effects in traditional toxicological parameters (LC50, EC50 or LOEC). One possibility is to include the post-exposure effects (e.g. delayed mortality) in the calculation of the toxicological parameters (e.g. LC50, EC50 or LOEC), given a more realistic assessment of the effects of toxicants. The inclusion of delayed effects may produce a reduction in the permissible or safe concentrations for aquatic environments, especially in toxicants with a rapid deleterious acute effect on animal physiology (as nitrite). In this way, traditional toxicological parameters might be related to exposure time effects (e.g. instantaneous LC50 or LOEC) and post-exposure time effects (e.g. delayed LC50 or LOEC for a given time). A similar toxicological parameter (as median post-exposure lethal time, peLT50) was proposed by Pascoe and Shazili (1986) to assess lethal time of cadmium to rainbow trout fry. Alternatively, bioassays can be conducted with a sufficient duration to observe both exposure and post-exposure effects (e.g. several hours or days after the end of exposure). Additionally, several toxicokinetic and toxicodynamic models have been proposed as a tool for mechanism-based
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ecotoxicology, allowing the simulation of the dynamic of toxicants in the whole organisms or to predict effects (Hickie et al., 1995; Ashauer et al., 2006, 2007; Unger et al., 2007; Ashauer and Brown 2008). As organism concentration is surrogated to the environmental concentration, these models can contribute to describe the dynamics of the deleterious effects and delayed mortality. Therefore, they can be a useful tool to assess pulse and delayed effects when only continuous exposure data are available.
5. Conclusions We conclude that the pulse duration and post-exposure were two factors that influenced in the lethal response of E. toletanus to nitrite, being the mortality at the end of the nitrite exposure lower than that after the end of the post-exposure period. Additionally, due to the high frequency of intermittent pollution in aquatic ecosystems, it would be necessary to incorporate the delayed mortality and other possible delayed effects into the traditional toxicological parameters and ecological risk assessment. This is especially relevant for very short-life toxicants (e.g. quickly oxidized, photo-degraded, etc.), that can stay in the water column for a few hours/days.
Acknowledgments This research was financed by the Spanish Ministry of Science and Technology (Research Project REN2001-1008) and Alcala University. Dr. A. Alonso was supported by the Council of CastillaLa Mancha Community and Alcala University (predoctoral grants). He is currently supported by the Spanish Ministry of Science and Innovation (Juan de la Cierva contract). Additionally, he has received a grant from the Wageningen Institute for Environment and Climate Research (WIMEK) to stay at the Aquatic Ecology and Water Quality Management Group (Wageningen University, The Netherlands). Our sincere gratitude to Pilar for correcting the English text and to Marcos de la Puente for his help with the taxonomic identification. Furthermore, we are grateful for two anonymous reviewers for their valuable comments to improve this manuscript. References Abel, P.D., Garner, S.M., 1986. Comparisons of median survival times and median lethal exposure times for Gammarus pulex exposed to cadmium, permethrin and cyanide. Water Res. 20, 579–582. Alonso, A., Camargo, J.A., 2004. Sub-lethal responses of the aquatic snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca) to unionized ammonia: a tolerant invading species. Fresen. Environ. Bull. 13, 607–615. Alonso, A., Camargo, J.A., 2006. Toxicity of nitrite to three species of freshwater invertebrates. Environ. Toxicol. 21, 90–94. Alonso, A., Camargo, J.A., 2008. Ameliorating effect of chloride on nitrite toxicity to freshwater invertebrates with different physiology: a comparative study between amphipods and planarians. Arch. Environ. Contam. Toxicol. 54, 259–265. American Public Health Association, 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. American Public Association, Washington, DC, USA.
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