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Ecotoxicological effects of citrus processing waste on earthworms, Lumbricus terrestris L.
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Ecotoxicological effects of citrus processing waste on earthworms, Lumbricus terrestris L. ⁎
Brighton M. Mvumia, , Willis Gwenzib, Munyaradzi G. Mhandua a
Department of Soil Science and Agricultural Engineering, Faculty of Agriculture, University of Zimbabwe, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe Biosystems and Environmental Engineering Research Group, Department of Soil Science and Agricultural Engineering, University of Zimbabwe, P.O. Box MP167, Mt. Pleasant, Harare, Zimbabwe b
A R T I C L E I N F O
A B S T R A C T
Keywords: Citrus processing waste Acute toxicity Soil chemical properties Earthworm fecundity Earthworm avoidance behaviour
Land disposal of agro-processing wastes containing potentially toxic compounds may lead to adverse ecotoxicological effects. Compared to agrochemicals, limited data are available on the ecotoxicological effects of biowastes such as those from citrus fruit processing. The objectives of the current study were:(1) to determine the effects of varying concentrations of citrus waste extract on adult mortality, fecundity and avoidance behaviour of the earthworm species Lumbricus terrestris under laboratory conditions; and (2) to determine earthworm abundance on contaminated and uncontaminated soils under field conditions. The laboratory experiment was a short-term acute toxicity test and it showed that the concentration of citrus extract had a significant (p < 0.05) negative effect on mortality, fecundity and avoidance behaviour. Exponential relationships (p < 0.001) were observed between citrus extract concentration and earthworm mortality (r2 = 0.98–0.99) and fecundity (r2 = 0.98), respectively. Tests showed that citrus waste-induced avoidance behaviour in earthworms changed by 44% to 100% compared to the control. Mean effect concentration (EC50) showed that earthworm avoidance behaviour (47%) was more sensitive to citrus waste concentrations than for number of cocoons (91%) and live earthworm counts (83%). Soil dumping of citrus waste significantly increased the concentration of mineral N, available N and exchange Ca, Mg and K but significantly reduced pH relative to the uncontaminated control. The shifts in chemical properties were about 1–2 orders of magnitude for P and Ca, double for K and approximately 30–60% for pH, N and Mg. Field data showed that the contaminated site had 80%significantly lower earthworm abundance than the uncontaminated control. Overall, the current study revealed that citrus waste has adverse ecotoxicological effects, which threatens the soil biota. Further studies should focus on elucidating the exact mechanisms and exploring the pesticidal effects of citrus extract solutions, particularly on meso- and macroinvertebrates. Investigations are also required to determine the persistence and mobility of the waste in soil and how to restore contaminated soils.
1. Introduction Citrus fruits are among some of the most popular agricultural products with an estimated global production of over 30 Mt per year (Mamma et al., 2008). Agro-processing, including citrus juice extraction is characterized by high waste-to-product ratio. Solid and liquid wastes from citrus processing pose significant public and environmental risks. Key disposal strategies for citrus processing solid wastes include landfilling, burning and incineration. Decomposition of organic wastes in landfills release methane, a potent greenhouse gas, while burning and incineration release carbon dioxide and particulate matter. In most developing countries including Zimbabwe, where sanitary landfills and incinerators are not available, land disposal is the most prevalent
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method for solid and liquid waste disposal. Land disposal of agrowastes potentially cause changes in soil chemical and physical properties, which may in turn have adverse impacts on soil biota and ecological functions. In addition, some agrowastes may contain toxic compounds that may directly affect soil ecology (e.g., Limonene in citrus wastes, Chubukov et al., 2015). These organic components could be released into the soil through leaching and decomposition and may pose direct ecotoxicological effects on soil fauna and flora. Moreover, citrus wastes may alter soil physical and chemical properties, and consequently ecological and hydrological functions (Khaleel et al., 1981). For example, oils and waxes produced during citrus waste decomposition form a coating layer around soil particles (Harris, 2013), and potentially induce water repellence. Water
Corresponding author. E-mail addresses: [email protected], [email protected] (B.M. Mvumi).
http://dx.doi.org/10.1016/j.indcrop.2017.09.003 Received 15 April 2017; Received in revised form 28 August 2017; Accepted 1 September 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Mvumi, B.M., Industrial Crops & Products (2017), http://dx.doi.org/10.1016/j.indcrop.2017.09.003
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Earthworms are also considered sensitive bioassay organisms widely used in standard ecotoxicity test procedures (e.g. OECD/OCED, 2015) and have been applied in several ecotoxocological studies (e.g., Svendsen et al., 2005; Lapied et al., 2010; Li et al., 2011). Specifically, Lumbricus terrestris L. has been used to investigate ecotoxocological effects of biocides (Svedsen et al., 2005) and nanomaterials (Lapied et al., 2010) and as an indicator of soil restoration (see review by Blouina et al., 2013). The current study sought to: (1) to determine the effects of varying concentrations of citrus waste extract on mortality, fecundity and avoidance behaviour of the earthworm species L. terrestris under laboratory conditions; and (2) to determine earthworm abundance on contaminated and uncontaminated soils under field conditions.
repellence restricts rainfall infiltration and affects soil-water relations, making it difficult for hydrophilic organisms such as the earthworms to survive. Apparently, earthworms lack a physiological mechanism to maintain a constant internal water content; hence their water content and activity are heavily dependent on the soil moisture (Kretzschmar and Bruchou, 1991). Earthworms need adequate moisture to help them respire through their skin. The optimum volumetric moisture levels in the soil for growth and development being 60–70% (Chauhan, 2014). Other studies have used relative humidity as an indicator of water stress and report that relative humidity lower than about 97% has adverse effects on earthworm growth, survival and fecundity (e.g. Holmstrup et al., 1998). In addition, citric acid may lower soil pH (Kimball, 1999), therefore affecting soil organisms and plants. Acidic pH conditions have negative effects on soil biota and carbon cycling processes (Alburquerque et al., 2012). Moreover, acidic conditions may lead to high solubility and bioavailability of toxic ions such as aluminium (Al) and manganese (Mn) which are highly toxic to many crops and the soil ecosystem (Gupta, 2011). This is particularly important for earthworms, considering that most of the species prefer a neutral pH although they can tolerate a pH of 5–8 (Edwards and Bohlen, 1996). These earlier studies suggest possible ecotoxicological effects of citrus wastes. Citrus waste extracts are lethal against mosquitoes, flies, termites and root-knot nematodes (Tsai, 2008). Tsai (2008) also showed that citrus peel extract is significantly nematicidal and nematostatic (paralysing effect) at 1:3 (w/v) dilution. Abolusoro et al. (2010) carried out similar experiments on the effectiveness of orange peel extract on rootknot nematodes, Melodogyne incognita. Their study revealed that orange peel extract was very effective at suppressing nematode population growth by inhibiting eggs from hatching and causing high juvenile mortality (92–96%) which compared well with the synthetic nematicide, carbofuran. These results suggest that these citrus waste products may have similar biocidal effects on many beneficial soil organisms, both micro- and macro-invertebrates. The exhibited nematicidal properties may be due to the presence of bioactive chemical compounds including; saponins, flavonoids and tannins in the citrus waste extract (Olabiyi, 2008). Murugan et al. (2012) showed the potential of citrus peel extracts in controlling different developmental stages of different mosquito species. A study by Oka et al. (2000) also showed the nematicidal effects of citrus oil extracts. Kimball (1999) carried out chemical analysis of the fresh citrus waste and deduced that it was highly acidic. However, few studies have investigated the ecotoxicological effects of land disposal of agrowastes such as citrus wastes. Earlier studies on citrus wastes have focussed on compositing (Van Heerden et al., 2002), production of animal feeds (Tripodo et al., 2004), biofuels (Lohrasbi et al., 2010) and application as an adsorbent in environmental remediation (Asgher and Bhatti, 2012). However, the effect on soil physico-chemical properties has received relatively little attention. Although citrus wastes have potential adverse effects on soil fauna and flora, little attention has been paid to their ecotoxicological effects on soil biota. Several ecotoxicological techniques such as bioassay and avoidance tests using earthworms have been developed but their application have largely been limited to the study of synthetic agrochemicals and heavy metals. Accordingly, there is limited information on the ecotoxicological effects of citrus wastes on macroinvertebrates such as earthworms. Earthworms play an important role in soil nutrient cycling, carrying out the process of mineralisation involving breaking down of complex organic molecules into nutrients that can be made available for plant uptake (Edwards and Bohlen, 1996; Chauhan, 2014). They improve soil aeration through burrowing into the soil thereby improving soil structure. However, the earthworms’ ability to perform these functions can be inhibited by exposure to toxic substances in the soil. Earthworms have been used as an important bioindicator organism for assessing sustainability of different environments (Paoletti, 1999; Shin and Kim, 2001; Gunadi and Edwards, 2003; Gibbs et al., 2009).
2. Materials and methods The study consisted of: (1) a laboratory simulated contamination of soils using five citrus extract concentration corresponding to 0, 25, 50, 75 and 100% w/w; and (2) a field experiment involving sampling of soils from contaminated and uncontaminated (control) plots. 2.1. Field experiment 2.1.1. Study site description The field experiment was conducted on a field at Mazowe Citrus Estate (MCE) in Mazowe district of Mashonaland Central province (30024′E, 29022′S), about 40 km North of Harare, the capital city of Zimbabwe. According to the Zimbabwe agroecological classification Mazowe lies in Natural Region II suitable for intensive agriculture. The climate of the area is tropical, characterized by warm average temperature (21 °C) and an average annual rainfall of about 864 mm, falling mainly in summer (October–March) (Mujere and Mazvimavi, 2012). Soils are predominantly in-situ red kaolinitic clay loam classified as Harare 2E.2 according to the Zimbabwe soil classification system (Nyamapfene, 1991), corresponding to Rhodic Kandiustalf according to USDA soil taxonomy (Almendros et al., 2001). Natural vegetation in the study area is miombo woodlands consisting of deciduous trees and a grass understorey. MCE processes approximately 40 000 Mt/yr of oranges into juice, generating about 20,000 Mt/yr of fresh citrus solid waste (Musoni et al., 2013), consisting predominantly of orange peels. The fresh solid waste is spread in 15-cm thick layers over an area of 0.4 ha. Considering an average moisture content of about 80% for orange wastes (M’hiri et al., 2015), this is equivalent to a high cumulative annual application rate of about 10 000 Mt/ha. Literature on recommended land application rates for citrus wastes is scarce, probably due to the fact that it is a rare practice considering the waste’s capacity to increase soil acidity. As expected, the current annual application at the waste dump was several orders of magnitude higher than maximum annual application rates for agricultural land (10 Mt//ha), and forest, rangeland and reclamation sites (150–250 Mt/ha/yr) (US EPA, 2000). The field experiment was conducted on two plots: Two sampling plots with the same soil type were identified within the study site: (1) an existing citrus processing waste dumpsite (subsequently referred to as the contaminated site) used for waste disposal for the past eight years and (2) an uncontaminated soil site (subsequently referred to as the control) about 1.5 km from the contaminated soil. Both the contaminated and the control soils were considered as the two treatments. The identification of contaminated and control sites was conducted with the assistance of officials from MCE and local people who had detailed information about the study site. 2.1.2. Soil chemical characterization Four replicate composite soil samples consisting of five random subsamples were collected from the top 30 cm using an auger in each of the contaminated and control sites for basic characterization. The samples 2
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were labelled and transported to the laboratory at University of Zimbabwe for chemical analysis. The samples were air-dried and passed through a 2-mm-diameter sieve. Thereafter, the soils were analysed for soil pH, soil organic carbon, mineral nitrogen, available phosphorus and exchangeable basic cations (Ca, Mg and K) and cation exchange capacity using standard methods (Okalebo et al., 1993). Soil pH was measured in 1:2 soil: 1 M calcium chloride suspension. The soil suspension was shaken for 1 h on a mechanical shaker and allowed to settle for 30 min. pH was them measured using a calibrated pH electrodes (model: Mettler Toledo). Exchangeable cations were determined by the 1.0 M ammonium acetate method. Ca and Mg, and K and Na were assayed using atomic absorption spectrophotometer and flame photometer respectively. Cation exchange capacity was determined as the summation of the exchangeable bases. Available phosphorus was determined by the Morgan soil test method (Wolf and Beegle, 2009), which uses sodium acetate buffered at pH 4.8 as extractant. The extract was filtered and the P in the filtered extract was extracted using resin. The concentration of P in solution was then determined by colorimetry using a UV visible spectrophotometer (Ketterings and Barney, 2010).
the top of each pot to get 100% cover to avoid excessive moisture loss from the soil. All the pots were kept under a thatch grass shed. Counts of live earthworms were conducted after 7 and 14 d of exposure (Abolusoro et al., 2010). The earthworms were placed back in the pots soon after the first count at 7 d.
2.2.3. Avoidance test The concentrations of citrus extract solutions were also used for the avoidance test. Self-draining rectangular trays (length: 30 cm, width: 20 cm and depth: 6 cm) were divided into four equal segments (circa 150 cm2) using square ceramic titles (area: 225 cm2, thickness: 4 mm). Soil treated with each of the four citrus extract solutions were packed into each segment. The trays were replicated four times. A total of 25 live adult earthworms were introduced at the centre of each tray, and the ceramic tiles separating the tiles were then removed. The earthworms were bought from a commercial producer in Harare, Zimbabwe and the identity verified by experts in the Department of Biological Sciences at the University of Zimbabwe. The trays were again covered with grass mulch as previously described to minimise evaporation and left to stay for 3 d (Garcia et al., 2008). Thereafter, the number of earthworms in each segment was counted.
2.1.3. Field earthworm abundance Earthworm counts were conducted on both contaminated and control plots, each measuring 10 × 10 m. Each plot was sub-divided into 1 m × 1 m sub-plots, giving 10 subplots for each treatment. Within each plot, five sub-plots were randomly selected for earthworm sampling. Each subplot was excavated to a depth of 30 cm, corresponding to the zone where earthworms are expected to be most prevalent (Sims and Gerard, 1985). The excavated soil was spread out on a plastic sheet for hand-sorting and the total number of earthworms in each sub-plot was counted and recorded (Sims and Gerard, 1985).
2.2.4. Fecundity assessment To determine the fecundity of the earthworms, after counting the number in each segment at 7 d, the adult earthworms were removed and transferred to corresponding fresh treatments on other trays. The soil in the first was then seived through thress sieve sizes (3.2 mm and 6.7 mm) and the cocoons counted. The number of cocoons in the fresh treatments was determined after another 7 d and 14-d cumulative number of cocoons recorded.
2.2. Laboratory experiment 2.3. Data analysis The general experimental procedures were adapted from the OECD guideline (2015).
Data were first tested for normality and homogeneity of variance using the Kolmogorov-Smirnov and Levene’s tests, respectively. The tests showed that numbers of cocoons and live earthworms, and avoidance test data violated ANOVA assumptions even after transformation. Moreover, earthworm avoidance data for each concentration are dependent on counts in other concentrations, thereby violating the assumption of independence of observations. Accordingly, the nonparametric Kruskal-Wallis test was used to determine the effect of citrus waste concentration on number of cocoons and live earthworms, while Friedman’s test was used for avoidance data. In cases where significant differences were observed, post-hoc tests were conducted to separate means. Probit analysis was used to determine dose-response relationships (i.e. median effect concentration, EC50) between citrus waste concentration and earthworm response. Probit analysis has been used to determine dose-response relationships in earlier ecotoxocological studies using earthworms as a bioassay organism (e.g., Loureiro et al., 2005; Zhou et al., 2007). All statistical analysis were done using SPSS® version 16 (SPSS Inc., 2007).
2.2.1. Treatment preparations The laboratory experiment consisted of acute toxicity, avoidance and fecundity tests using Lumbricus terrestris L., positively identified at the Department of Biological Sciences at University of Zimbabwe. The choice of L. terrestris as a bioassay species was based on results of determination of field abundance indicating that this was the only species observed. Five concentrations of aqueous citrus extract solutions were prepared from fresh citrus Citrus sinensis L. waste (orange peels) collected from the waste dump site at MCE. The orange peels were crushed using a mortar and pestle. Aqueous citrus extract solutions of various concentrations were prepared by mixing known masses of the crushed fresh orange peels and volumes of deionized water. The following solutions, which constituted the treatments, were prepared on a weight/ volume (w/v) basis: 0% consisting of deionized water (control), 0.25:1 (25%), 0.5: 1 (50%), 0.75: 1 (75%) and 1: 1 (100% citrus extract). The solutions were left to stand for 2 d, after which they were filtered and used in the experiment.
3. Results
2.2.2. Acute toxicity test For the acute toxicity test, clean bulk soil samples, enough to fill four replicates of 1-L earthen pots (measuring 23 cm top diameter and 15 cm bottom diameter and 23 cm depth) per treatment were prepared and the five citrus extract solutions applied to the pots separately. The soil was thoroughly mixed with the citrus extract solutions, and then loosely packed in the 1-L pots. In each pot, ten mature, live earthworms (OECD, 2015) of length 5.5 ± 0.5 cm (mean ± standard error of the mean), were introduced in each pot. The earthworms were then covered with 5 cm of the corresponding treated soil. A dry mulch of couch grass, Cynodon dactylon (L.) Pers. cut by a lawn mower, was applied at
3.1. Soil chemical properties Analysis of selected soil chemical properties showed that the contaminated soil was acidic (4.8), while the uncontaminated soil was slightly alkaline (7.9) (Table 1). Citrus waste contaminated soil had significantly higher mineral N, available P and exchangeable Ca, Mg and K than the uncontaminated control. The shifts in chemical properties were about 1–2 orders of magnitude for P and Ca, two-fold for K and approximately 30–60% for pH, N and Mg. 3
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Table 1 Selected soil chemical properties for the citrus waste contaminated site and the uncontaminated control. Means with different letters are significantly different at probability p = 0.05.
Contaminated Uncontaminated
pH
Mineral N (mg/kg)
Available P, resin extract (mg/kg)
Potassium (mg/kg)
Calcium (mg/kg)
Magnesium (mg/kg)
4.8a 7.9b
52a 41b
151a 8b
11.0a 5.2b
407.4a 37.6b
55.3a 33.7b
Fig. 1. Mean number of live adult earthworms (L. terrestris) remaining out of the initial 10 per pot after the 7 and 14-d test periods (n = 4). Error bars denote standard error of the mean. Means for each concentration with different letters within each test period are significantly different at probability, p < 0.001.
3.2. Field earthworm abundance A t-test comparison of field counts showed that the citrus waste contaminated site had approximately 80% significantly (p < 0.001) lower earthworm abundance (0.8 ± 0.37 per m2) than the uncontaminated control (9.8 ± 0.81 per m2). In summary, results of both laboratory simulation and field measurements demonstrate that citrus waste extract had an adverse effect on earthworms. 3.3. Laboratory experiment 3.3.1. Acute toxicity effects on adult earthworms Citrus waste extract had significant (p < 0.05) biocidal effects on adult earthworms (Fig. 1). Significant (p < 0.001) reductions in the number of live adult earthworms were observed among all concentrations except 25% and 54%, which were similar. The number of live earthworms in the 100% citrus extract was about two times lower than that of the control. The number of live adult earthworms showed a strong exponential decline with increasing citrus extract concentration after 7 (y = 9.69e−0.006x, r2 = 0.96 d). A similar trend was observed after 14 d. However, for each citrus extract concentration, the numbers of live adults after 7- and 14-d exposure periods were similar.Probit analysis of data on live earthworms yielded mean effect concentration (EC50) of 83% citrus waste concentrations.
Fig. 2. Mean number of earthworms (L. terrestris) in compartments after a 2-d exposure to soils contaminated with various concentrations of citrus extract (n = 4). Error bars denote standard error of the mean. Bars with different letters are significantly different at p < 0.001.
observed in the avoidance test decreased exponentially with citrus waste concentration (y = 16.0 e−0.037x, r2 = 0.96, p < 0.001). The results clearly showed that avoidance was directly related to citrus extract concentration. The EC50 for earthworm avoidance behaviour was EC50 47% citrus waste concentrations.
3.3.2. Avoidance behaviour The number of earthworms in each compartment significantly (p < 0.05) declined with increasing concentration of citrus extract (Fig. 2). Earthworm counts varied significantly among all citrus extract concentrations, and declined significantly (p < 0.05) with increasing concentrations. Citrus waste-induced avoidance behaviour in earthworms changed by 44% to 100% compared to the control, with the earthworms completely avoiding the compartment with 100% citrus concentration. Compartments with 25% and 50% citrus extract concentrations accounted for 32% and 9% of the total number of earthworms introduced, respectively. The number of earthworm counts
3.3.3. Effects on fecundity The number of earthworm cocoons was used as an indicator of fecundity. Significant reductions (p < 0.002) in the number of earthworm cocoons were observed at different concentrations of citrus extract compared to the control (Fig. 3). An exception was the 50 and 75% concentrations which had similar numbers of cocoons. The number of earthworm cocoons (EC) exponentially declined with increasing concentration of citrus waste extract (C) (p < 0.001). The corresponding 4
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which include tannins, flavonoids and saponins which they showed to have biocidal effects on the root knot nematode, Melodogyne incognita. Davidson (2001) studied the effects of citrus extract on fleas and reported that D-limonene and linalool found in the citrus extract were responsible for the mortality observed on flea adults and larvae. D-limonene, an ingredient of orange oil has been demonstrated to be fatal against a variety of organisms which include ants, mosquitoes, flies, crickets and termites. The oil dissolves the insects’ exoskeleton, destroying the insect’s cell membranes. The loss of the exoskeleton in insects and other soil organisms consequently results in dying from excessive loss of water and proteins (Harris, 2013). The extraction and subsequent processing of citrus products also entail use of chemical additives. As shown by the data for the selected chemical properties, these chemical may accumulate in citrus waste and could have adverse impacts on chemical properties of soils. Therefore, besides the direct effects of chemical compounds originating from citrus, there is a possibility that these changes in habitat chemical properties induced by chemical additives may contribute to mortality and avoidance behaviour evident in both laboratory simulation and field studies. The increased avoidance behaviour of earthworms with increasing concentration of citrus extract observed could be ascribed to the strong olfactory senses that the organisms use to identify food and to avoid poisonous environments (Tsai, 2008). This implies that as the concentration of the citrus extract increased, the odour of the active compounds also increased and the earthworms avoided them. Iordache et al. (2010) also carried out avoidance tests on earthworms using inorganic fertilisers and obtained almost similar results. They attributed such behaviour to the earthworms’ olfactory capabilities. Therefore our results suggest that earthworms preferentially avoid soils contaminated with citrus waste. Citrus contamination also had significant negative effects on the fecundity of earthworms. It reduced the number of cocoons produced by earthworms, probably also contributing to low earthworm populations in the soil. This implies that soils contaminated with citrus waste are not a conducive habitat for earthworms. Kirkpatric (2003) studied optimum conditions necessary for earthworm survival and observed that under ‘stressful’ conditions, earthworms lose their clitellum (glandular swelling on earthworms). The clitellum is involved in reproduction (Donahue, 2003). This is the most probable cause in the decrease in fecundity and also the decrease in population caused by the citrus extract. The low earthworm population in the contaminated plots could also be ascribed to the fact that if earthworms have to “fight” against the limiting factors such as the contaminants or the low pH, they use their energy reserves for that, instead of the biological processes such as reproduction and growth according to the theory of dynamic energy budget (Kooijman, 2009). Butt (1991) showed that with food supply L. terrestris, fecundity decreased from 1.9 cocoon per month at 18–21 °C to less than 1 cocoon per month as temperature rose to 22 °C. Mean ambient temperatures during the current study were more than 25 °C which would negatively affect the earthworms. The drop in pH from 7.9 for the untreated control to 4.8 for the citrus waste contaminated soils was attributed to organic acids (e.g. citric and limoninic acids) derived from citrus wastes (Kimball, 1999). The acidic pH was below the near-neutral (6.5–7.5) considered optimum for earthworms (Edwards and Bohlen, 1996), and could partly account for the reduced fecundity and survival observed in citrus waste contaminated soils. The profound adverse effects on soil pH could be attributed to the very high citrus waste application rates (i.e., 15 cmlayer equivalent to 10000 Mt/ha/year). Assuming the acidic soil pH is one of the key factors accounting for the adverse impacts of citrus waste on soil biota, two options exist to minimize the ecological impacts. First, future disposal should reduce annual application rates from the current 15 cm or 10000 Mt/ha/year by about one to three orders of magnitude to attain typical maximum recommended rates (150–250 Mt/ha/year) for bio-solid land application (USEPA, 2000).
Fig. 3. Mean number of earthworm (L. terrestris) cocoons after 7- and 14-d exposure periods (n = 4) for an initial 10 earthworms per pot. Error bars denote standard error of the mean. Means with different letters are significantly different at probability, p < 0.001. Note that cocoons were recorded and discarded after the first 7 d and thereafter earthworms placed back for another 7 d to make a cumulative 14 d.
exponential decay functions for the 7- and 14-d exposure periods were: EC7-d = 63.1e0.49C (r2 = 0.98) and EC14-d = 70.5e0.43C (r2 = 0.99), respectively. Across the various citrus extract concentrations, the number of cocoons for the 7-d exposure period was generally higher than that for the 14-d period. The EC50 for numbers of cocoon was approximately 91% citrus waste concentrations.
4. Discussion The results show that the citrus waste has significant effects on earthworm abundance in soils. Concentrations as low as 25% showed mortality rates of at least 20% and almost 50% mortality at concentrations around 75%. The longer the exposure period, the higher the mortality with mortality increasing by almost 90% at the highest level of contamination with citrus waste after 14 d. Sampling at the Mazowe dumpsite showed that citrus waste has severe effects on the populations of earthworms, with populations at the uncontaminated sites at least six times greater than those at the contaminated site. The general low numbers of earthworms record at both sites could attributed to the fact that the study was conducted towards the end of the wet season. Gelsomino et al. (2010) highlighted that the alternative use of citrus processing waste as soil conditioner in agriculture has been investigated without encouraging results. They pointed out that repeated application of large amounts of citrus waste have shown evidence of significant limitations. Citrus waste application to agricultural soils has shown notable negative effects on both soil chemical properties and crop yields (Bellingo et al., 2005). Loss of soil quality due to elevated metal ions leads to decreased yields due to decreased pH, loss of basic cations, inhibition of nitrification, promotion of certain diseases and fixation of major anion nutrients (Gupta, 2011). Some of these adverse effects of citrus waste result in decrease soil faunal population. Survival and reproduction of earthworms decreased with increasing concentration of citrus waste extract. Avoidance also increased with increase in concentration; with no earthworms found in the portion with 100% citrus extract concentration. Lower EC50 (47%) was observed for avoidance behaviour than numbers of cocoons (91%) and live earthworms (83%), suggesting that earthworm avoidance behaviour was more sensitive to citrus waste concentration than fecundity and survival. The effects on earthworm survival were probably due to the citrus waste containing toxic compounds that have serious biocidal effects on earthworms. It was not the objective of this study to find the chemical composition of the citrus waste but previous studies by Abolusoro et al. (2010) suggested that citrus peels contain bioactive organic compounds 5
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Iordache, M., Gaica, I., Borza, I., Dica, D., 2010. Ecotoxicological Assessment of the Effects of Some New Organic Mineral Fertilisers on Eisenia Foetida Earthworms. University of agricultural Science and Veterinary Medicine of Banat, Tinisoara. Ketterings, J., Barney, A.B., 2010. Nutrient Management Program. Cornell University Cooperative Extension. http://nmsp.cals.cornell.edu. Khaleel, R., Reddy, K.R., Overcash, M.R., 1981. Changes in soil physical properties due to organic waste applications: a review. J. Environ. Qual. 10 (2), 133–141. Kimball, D.A., 1999. Analyses of brix, soluble solids, ccids, oils, and pulp. Citrus Processing. Springer, pp. 191–246. Kirkpatric, T., 2003. Investigating the optimum laboratory conditions for rearing of earthworms for vermiculture. J. Soil Biol. 18, 340. Kooijman, S.A.L.M., 2009. Dynamic Energy Budget Theory for Metabolic Organisation. Cambridge University Press, Cambridge xvi + 490 pp. Kretzschmar, A., Bruchou, C., 1991. Weight response to the soil water potential of the earthworm Aporrectodea longa. Biol. Fertil. Soils 12, 209–212. Lapied, E., Moudilou, E., Exbrayat, J.M., Oughton, H., Joner, E.J., 2010. Silver nanoparticle exposure causes apoptotic response in the earthworm Lumbricus terrestris (Oligochaeta). Nanomed 5, 975–984. Li, D., Hockaday, W.C., Masiello, C.A., Alvarez, P.J.J., 2011. Earthworm avoidance of biochar can be mitigated by wetting. Soil Biol. Biochem. 43, 1732–1737. Lohrasbi, M., Pourbafrani, M., Niklasson, C., Taherzadeh, M.J., 2010. Process design and economic analysis of a citrus waste biorefinery with biofuels and limonene as products. Bioresour. Technol. 101, 7382–7388. Loureiro, S., Soares, A.M., Nogueira, A.J., 2005. Terrestrial avoidance behaviour tests as screening tool to assess soil contamination. Environ. Pollut. 138 (1), 121–131. M’hiri, N., Ioannou, I., Ghoul, M., Boudhrioua, N.M., 2015. Proximate chemical composition of orange peel and variation of phenols and antioxidant activity during convective air drying. J. New Sci. 9, 881–890. Martin, M.A., Siles, J.A., Chica, A.F., Martina, A., 2010. Biomethanization of orange peel waste. Bioresour. Technol. 101, 8993–8999. Mujere, N., Mazvimavi, D., 2012. Impact of climate change on reservoir reliability. Afr. Crop Sci. J. 20, 545–551. Murugan, K., Mahesh, K.P., Kovendan, K., Amerasan, D., Subrmaniam, J., Hwang, J.S., 2012. Larvicidal, pupicidal, repellent and adulticidal activity of Citrus sinensis orange peel extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae). Parasitol. Res. 111, 1757–1769. Musoni, S., Madanhire, I., Mugwindiri, K., 2013. Developing a cleaner production system for citrus processing: a case study of a developing country. Int. J. Appl. Innov. Eng. Manage. 2, 1–10. Nguyen, H., 2012. Biogas Production from Solvent Pretreated Orange Peel. Department of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden. Nyamapfene, K.W., 1991. Soils of Zimbabwe. Nehanda Publ (Pvt), Harare.
This can be achieved by spreading the citrus waste over a larger area. Second, soil acidity on existing citrus waste contaminated soils can be neutralized through application of lime and/or other readily available low-cost alkaline amendments such as coal ash and ash from combustion of solid waste. In this regard, application of lime and/or other alkaline amendments could aim to achieve a pH of 6.5, which could in turn, promote long-term ecological recovery. Although the current study used earthworms as a bioassay organism, the results may have implications on other soil macro-invertebrates and possibly other soil organisms. The observed mortality and avoidance behaviour of the earthworm imply that citrus wastes may undergo slow decomposition process due to their potentially toxic effects on soil organisms. This has implications on the fate of such wastes when disposed of in waste repositories such as landfills, land application and their use in waste-to-energy recycling programs. For instance, in biogas digesters, putative changes in digestate pH and microbial communities caused by use of citrus wastes may adversely affect biogas production. Indeed, several studies investigating biogas production from citrus waste confirm that the presence of toxic compounds such as limonene adversely affect the use of such wastes as biogas feedstock if no pre-treatment is done (Martin et al., 2010; Nguyen, 2012; Wikandari et al., 2014). 5. Conclusion Citrus solid waste had adverse effects on the survival, fecundity, and avoidance behaviour of earthworms. Fecundity, survival and number of earthworms in avoidance test declined exponentially with increasing citrus waste concentration. However, mean effect concentration (EC50) showed that earthworm avoidance behaviour was more sensitive to citrus waste concentrations than fecundity and survival. These adverse could be attributed to acidic pH and potentially toxic organic compounds in citrus waste such as limonene. Our results demonstrate that soil biota can be negatively affected by excessive exposure to citrus processing waste, pointing to the need for caution when disposing such wastes on land. Long-term solutions may include proper citrus processing waste disposal practices such as engineered landfills and conversion to value-added products such as stockfeed are required to prevent the adverse ecological impacts. Possible options to minimize the ecological impacts of citrus solid waste include: (1) reducing current application rates to maximum internationally recommended rates for land application of bio-solids by spreading the citrus waste over a large area; and (2) neutralizing soil acidity through application of lime and/or other readily available low-cost alkaline materials such as coal ash and ash from combustion of solid waste. Further research on already contaminated sites could investigate phytoremediation with acid-tolerant plant species such as vetiver grass and star grass. Acknowledgements We are grateful to the Mazowe Citrus Estate (MCE) for allowing us to conduct this study on their premises. We acknowledge technical and logistical support provided by the staff in the Department of Soil Science and Agricultural Engineering, University of Zimbabwe (UZ). The expertise provided by staff in the Department of Biological Sciences, UZ in correctly identifying the earthworm species is greatly appreciated. References Abolusoro, S.A., Oyedunmade, E.A., Olabiyi, T.I., 2010. Evaluation of sweet orange peel aqueous extract (Citrus sinensis) as root-knot nematode suppressant. Agro-Sci. J. Trop. Agric. Food Environ. Ext. 9, 170–175. Alburquerque, J.A., de la Fuente, C., Bernal, M.P., 2012. Chemical properties of anaerobic digestates affecting C and N dynamics in amended soils. Agric. Ecosyst. Environ. 160, 15–22. Almendros, G., Giampaolo, S., Pardo, M.T., 2001. Laboratory appraisal of carbon
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antiparasitic compounds ivermectin and fenbendazole. Soil Biol. Biochem. 37, 927–936. Tripodo, M.M., Lanuzza, F., Micali, G., Coppolino, R., Nucita, F., 2004. Citrus waste recovery: a new environmentally friendly procedure to obtain animal feed. Bioresour. Technol. 91 (2), 111–115. Tsai, B.Y., 2008. Effect of peels of lemon, orange, and grapefruit against Meloidogyne incognita. Plant Pathol. Bull. 17, 195–201. US EPA (U.S. Environmental Protection Agency), 2000. Biosolids Technology Fact Sheet: Land Application of Biosolids. Environmental Protection Agency, Washington D.C EPA 832-F-00-064. U.S. Van Heerden, I., Cronjé, C., Swart, S.H., Kotzé, J.M., 2002. Microbial, chemical and physical aspects of citrus waste composting. Bioresour. Technol. 81 (1), 71–76. Wikandari, R., Millati, R., Cahyanto, M.N., Taherzadeh, M.I., 2014. Biogas production from citrus waste by membrane bioreactor. Membranes 4, 596–607. Wolf, A., Beegle, D., 2009. Recommended soil tests for macro and micronutrients. Chapter 5. Recommended soil testing procedures for the northeastern United States, third ed. Northeastern Regional Cooperative Bulletin, No. 493, pp. 40––47. Zhou, S.P., Duan, C.Q., Hui, F.U., Chen, Y.H., Wang, X.H., Yu, Z.F., 2007. Toxicity assessment for chlorpyrifos-contaminated soil with three different earthworm test methods. J. Environ. Sci. 19 (7), 854–858.
OECD/OCED, 2015. Guideline for the Testing of Chemicals. Earthworm Reproduction Test (Eisenia Fetida/Eisenia Andrei). Draft updated TG 222. Draft, 12 June 2015. 19pp. Oka, Y., Nacar, S., Putievsky, E., Ravid, U., Yaniv, L., Spiegel, Y., 2000. Nematicidal activity of essential oils and their components against the root-knot nematode. Phytopathology 90, 710–715. Okalebo, J.R., Gathua, K.W., Woomer, P.I., 1993. Laboratory Methods of Soil and Plant Analysis: A Working Manual. Tropical Soil Biology and Fertility Programme, Nairobi. Olabiyi, T.I., 2008. Pathogenicity study and nematoxic properties of some plant extracts on the root-knot nematode pest of tomato, Lycopersicon esculentum. Plant Pathol. J. 7 (1), 45–49. Paoletti, M.G., 1999. The role of earthworms for assessment of sustainability and as bioindicators. Agric. Ecosyst. Environ. 74, 137–155. SPSS Inc, 2007. SPSS for Windows, Version 16.0. SPSS Inc., Chicago Released 2007. Shin, K.H., Kim, K.W., 2001. Ecotoxicity monitoring of hydrocarbon- contaminated soil using earthworm (Eisenia foetida). Environ. Monit. Assess. 70, 93–103. Sims, R.W., Gerard, B.M., 1985. Earthworms: Keys and Notes for the Identification and Study of the Species, vol. 31 The Linnean Society of London, London. Svendsen, T.S., Hansen, P.E., Sommer, C., Martinussen, T., Grønvold, J., Holter, P., 2005. Life history characteristics of Lumbricus terrestris and effects of the veterinary
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Ecotoxicological effects of citrus processing waste on earthworms, Lumbricus terrestris L. ⁎
Brighton M. Mvumia, , Willis Gwenzib, Munyaradzi G. Mhandua a
Department of Soil Science and Agricultural Engineering, Faculty of Agriculture, University of Zimbabwe, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe Biosystems and Environmental Engineering Research Group, Department of Soil Science and Agricultural Engineering, University of Zimbabwe, P.O. Box MP167, Mt. Pleasant, Harare, Zimbabwe b
A R T I C L E I N F O
A B S T R A C T
Keywords: Citrus processing waste Acute toxicity Soil chemical properties Earthworm fecundity Earthworm avoidance behaviour
Land disposal of agro-processing wastes containing potentially toxic compounds may lead to adverse ecotoxicological effects. Compared to agrochemicals, limited data are available on the ecotoxicological effects of biowastes such as those from citrus fruit processing. The objectives of the current study were:(1) to determine the effects of varying concentrations of citrus waste extract on adult mortality, fecundity and avoidance behaviour of the earthworm species Lumbricus terrestris under laboratory conditions; and (2) to determine earthworm abundance on contaminated and uncontaminated soils under field conditions. The laboratory experiment was a short-term acute toxicity test and it showed that the concentration of citrus extract had a significant (p < 0.05) negative effect on mortality, fecundity and avoidance behaviour. Exponential relationships (p < 0.001) were observed between citrus extract concentration and earthworm mortality (r2 = 0.98–0.99) and fecundity (r2 = 0.98), respectively. Tests showed that citrus waste-induced avoidance behaviour in earthworms changed by 44% to 100% compared to the control. Mean effect concentration (EC50) showed that earthworm avoidance behaviour (47%) was more sensitive to citrus waste concentrations than for number of cocoons (91%) and live earthworm counts (83%). Soil dumping of citrus waste significantly increased the concentration of mineral N, available N and exchange Ca, Mg and K but significantly reduced pH relative to the uncontaminated control. The shifts in chemical properties were about 1–2 orders of magnitude for P and Ca, double for K and approximately 30–60% for pH, N and Mg. Field data showed that the contaminated site had 80%significantly lower earthworm abundance than the uncontaminated control. Overall, the current study revealed that citrus waste has adverse ecotoxicological effects, which threatens the soil biota. Further studies should focus on elucidating the exact mechanisms and exploring the pesticidal effects of citrus extract solutions, particularly on meso- and macroinvertebrates. Investigations are also required to determine the persistence and mobility of the waste in soil and how to restore contaminated soils.
1. Introduction Citrus fruits are among some of the most popular agricultural products with an estimated global production of over 30 Mt per year (Mamma et al., 2008). Agro-processing, including citrus juice extraction is characterized by high waste-to-product ratio. Solid and liquid wastes from citrus processing pose significant public and environmental risks. Key disposal strategies for citrus processing solid wastes include landfilling, burning and incineration. Decomposition of organic wastes in landfills release methane, a potent greenhouse gas, while burning and incineration release carbon dioxide and particulate matter. In most developing countries including Zimbabwe, where sanitary landfills and incinerators are not available, land disposal is the most prevalent
⁎
method for solid and liquid waste disposal. Land disposal of agrowastes potentially cause changes in soil chemical and physical properties, which may in turn have adverse impacts on soil biota and ecological functions. In addition, some agrowastes may contain toxic compounds that may directly affect soil ecology (e.g., Limonene in citrus wastes, Chubukov et al., 2015). These organic components could be released into the soil through leaching and decomposition and may pose direct ecotoxicological effects on soil fauna and flora. Moreover, citrus wastes may alter soil physical and chemical properties, and consequently ecological and hydrological functions (Khaleel et al., 1981). For example, oils and waxes produced during citrus waste decomposition form a coating layer around soil particles (Harris, 2013), and potentially induce water repellence. Water
Corresponding author. E-mail addresses: [email protected], [email protected] (B.M. Mvumi).
http://dx.doi.org/10.1016/j.indcrop.2017.09.003 Received 15 April 2017; Received in revised form 28 August 2017; Accepted 1 September 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Mvumi, B.M., Industrial Crops & Products (2017), http://dx.doi.org/10.1016/j.indcrop.2017.09.003
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Earthworms are also considered sensitive bioassay organisms widely used in standard ecotoxicity test procedures (e.g. OECD/OCED, 2015) and have been applied in several ecotoxocological studies (e.g., Svendsen et al., 2005; Lapied et al., 2010; Li et al., 2011). Specifically, Lumbricus terrestris L. has been used to investigate ecotoxocological effects of biocides (Svedsen et al., 2005) and nanomaterials (Lapied et al., 2010) and as an indicator of soil restoration (see review by Blouina et al., 2013). The current study sought to: (1) to determine the effects of varying concentrations of citrus waste extract on mortality, fecundity and avoidance behaviour of the earthworm species L. terrestris under laboratory conditions; and (2) to determine earthworm abundance on contaminated and uncontaminated soils under field conditions.
repellence restricts rainfall infiltration and affects soil-water relations, making it difficult for hydrophilic organisms such as the earthworms to survive. Apparently, earthworms lack a physiological mechanism to maintain a constant internal water content; hence their water content and activity are heavily dependent on the soil moisture (Kretzschmar and Bruchou, 1991). Earthworms need adequate moisture to help them respire through their skin. The optimum volumetric moisture levels in the soil for growth and development being 60–70% (Chauhan, 2014). Other studies have used relative humidity as an indicator of water stress and report that relative humidity lower than about 97% has adverse effects on earthworm growth, survival and fecundity (e.g. Holmstrup et al., 1998). In addition, citric acid may lower soil pH (Kimball, 1999), therefore affecting soil organisms and plants. Acidic pH conditions have negative effects on soil biota and carbon cycling processes (Alburquerque et al., 2012). Moreover, acidic conditions may lead to high solubility and bioavailability of toxic ions such as aluminium (Al) and manganese (Mn) which are highly toxic to many crops and the soil ecosystem (Gupta, 2011). This is particularly important for earthworms, considering that most of the species prefer a neutral pH although they can tolerate a pH of 5–8 (Edwards and Bohlen, 1996). These earlier studies suggest possible ecotoxicological effects of citrus wastes. Citrus waste extracts are lethal against mosquitoes, flies, termites and root-knot nematodes (Tsai, 2008). Tsai (2008) also showed that citrus peel extract is significantly nematicidal and nematostatic (paralysing effect) at 1:3 (w/v) dilution. Abolusoro et al. (2010) carried out similar experiments on the effectiveness of orange peel extract on rootknot nematodes, Melodogyne incognita. Their study revealed that orange peel extract was very effective at suppressing nematode population growth by inhibiting eggs from hatching and causing high juvenile mortality (92–96%) which compared well with the synthetic nematicide, carbofuran. These results suggest that these citrus waste products may have similar biocidal effects on many beneficial soil organisms, both micro- and macro-invertebrates. The exhibited nematicidal properties may be due to the presence of bioactive chemical compounds including; saponins, flavonoids and tannins in the citrus waste extract (Olabiyi, 2008). Murugan et al. (2012) showed the potential of citrus peel extracts in controlling different developmental stages of different mosquito species. A study by Oka et al. (2000) also showed the nematicidal effects of citrus oil extracts. Kimball (1999) carried out chemical analysis of the fresh citrus waste and deduced that it was highly acidic. However, few studies have investigated the ecotoxicological effects of land disposal of agrowastes such as citrus wastes. Earlier studies on citrus wastes have focussed on compositing (Van Heerden et al., 2002), production of animal feeds (Tripodo et al., 2004), biofuels (Lohrasbi et al., 2010) and application as an adsorbent in environmental remediation (Asgher and Bhatti, 2012). However, the effect on soil physico-chemical properties has received relatively little attention. Although citrus wastes have potential adverse effects on soil fauna and flora, little attention has been paid to their ecotoxicological effects on soil biota. Several ecotoxicological techniques such as bioassay and avoidance tests using earthworms have been developed but their application have largely been limited to the study of synthetic agrochemicals and heavy metals. Accordingly, there is limited information on the ecotoxicological effects of citrus wastes on macroinvertebrates such as earthworms. Earthworms play an important role in soil nutrient cycling, carrying out the process of mineralisation involving breaking down of complex organic molecules into nutrients that can be made available for plant uptake (Edwards and Bohlen, 1996; Chauhan, 2014). They improve soil aeration through burrowing into the soil thereby improving soil structure. However, the earthworms’ ability to perform these functions can be inhibited by exposure to toxic substances in the soil. Earthworms have been used as an important bioindicator organism for assessing sustainability of different environments (Paoletti, 1999; Shin and Kim, 2001; Gunadi and Edwards, 2003; Gibbs et al., 2009).
2. Materials and methods The study consisted of: (1) a laboratory simulated contamination of soils using five citrus extract concentration corresponding to 0, 25, 50, 75 and 100% w/w; and (2) a field experiment involving sampling of soils from contaminated and uncontaminated (control) plots. 2.1. Field experiment 2.1.1. Study site description The field experiment was conducted on a field at Mazowe Citrus Estate (MCE) in Mazowe district of Mashonaland Central province (30024′E, 29022′S), about 40 km North of Harare, the capital city of Zimbabwe. According to the Zimbabwe agroecological classification Mazowe lies in Natural Region II suitable for intensive agriculture. The climate of the area is tropical, characterized by warm average temperature (21 °C) and an average annual rainfall of about 864 mm, falling mainly in summer (October–March) (Mujere and Mazvimavi, 2012). Soils are predominantly in-situ red kaolinitic clay loam classified as Harare 2E.2 according to the Zimbabwe soil classification system (Nyamapfene, 1991), corresponding to Rhodic Kandiustalf according to USDA soil taxonomy (Almendros et al., 2001). Natural vegetation in the study area is miombo woodlands consisting of deciduous trees and a grass understorey. MCE processes approximately 40 000 Mt/yr of oranges into juice, generating about 20,000 Mt/yr of fresh citrus solid waste (Musoni et al., 2013), consisting predominantly of orange peels. The fresh solid waste is spread in 15-cm thick layers over an area of 0.4 ha. Considering an average moisture content of about 80% for orange wastes (M’hiri et al., 2015), this is equivalent to a high cumulative annual application rate of about 10 000 Mt/ha. Literature on recommended land application rates for citrus wastes is scarce, probably due to the fact that it is a rare practice considering the waste’s capacity to increase soil acidity. As expected, the current annual application at the waste dump was several orders of magnitude higher than maximum annual application rates for agricultural land (10 Mt//ha), and forest, rangeland and reclamation sites (150–250 Mt/ha/yr) (US EPA, 2000). The field experiment was conducted on two plots: Two sampling plots with the same soil type were identified within the study site: (1) an existing citrus processing waste dumpsite (subsequently referred to as the contaminated site) used for waste disposal for the past eight years and (2) an uncontaminated soil site (subsequently referred to as the control) about 1.5 km from the contaminated soil. Both the contaminated and the control soils were considered as the two treatments. The identification of contaminated and control sites was conducted with the assistance of officials from MCE and local people who had detailed information about the study site. 2.1.2. Soil chemical characterization Four replicate composite soil samples consisting of five random subsamples were collected from the top 30 cm using an auger in each of the contaminated and control sites for basic characterization. The samples 2
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were labelled and transported to the laboratory at University of Zimbabwe for chemical analysis. The samples were air-dried and passed through a 2-mm-diameter sieve. Thereafter, the soils were analysed for soil pH, soil organic carbon, mineral nitrogen, available phosphorus and exchangeable basic cations (Ca, Mg and K) and cation exchange capacity using standard methods (Okalebo et al., 1993). Soil pH was measured in 1:2 soil: 1 M calcium chloride suspension. The soil suspension was shaken for 1 h on a mechanical shaker and allowed to settle for 30 min. pH was them measured using a calibrated pH electrodes (model: Mettler Toledo). Exchangeable cations were determined by the 1.0 M ammonium acetate method. Ca and Mg, and K and Na were assayed using atomic absorption spectrophotometer and flame photometer respectively. Cation exchange capacity was determined as the summation of the exchangeable bases. Available phosphorus was determined by the Morgan soil test method (Wolf and Beegle, 2009), which uses sodium acetate buffered at pH 4.8 as extractant. The extract was filtered and the P in the filtered extract was extracted using resin. The concentration of P in solution was then determined by colorimetry using a UV visible spectrophotometer (Ketterings and Barney, 2010).
the top of each pot to get 100% cover to avoid excessive moisture loss from the soil. All the pots were kept under a thatch grass shed. Counts of live earthworms were conducted after 7 and 14 d of exposure (Abolusoro et al., 2010). The earthworms were placed back in the pots soon after the first count at 7 d.
2.2.3. Avoidance test The concentrations of citrus extract solutions were also used for the avoidance test. Self-draining rectangular trays (length: 30 cm, width: 20 cm and depth: 6 cm) were divided into four equal segments (circa 150 cm2) using square ceramic titles (area: 225 cm2, thickness: 4 mm). Soil treated with each of the four citrus extract solutions were packed into each segment. The trays were replicated four times. A total of 25 live adult earthworms were introduced at the centre of each tray, and the ceramic tiles separating the tiles were then removed. The earthworms were bought from a commercial producer in Harare, Zimbabwe and the identity verified by experts in the Department of Biological Sciences at the University of Zimbabwe. The trays were again covered with grass mulch as previously described to minimise evaporation and left to stay for 3 d (Garcia et al., 2008). Thereafter, the number of earthworms in each segment was counted.
2.1.3. Field earthworm abundance Earthworm counts were conducted on both contaminated and control plots, each measuring 10 × 10 m. Each plot was sub-divided into 1 m × 1 m sub-plots, giving 10 subplots for each treatment. Within each plot, five sub-plots were randomly selected for earthworm sampling. Each subplot was excavated to a depth of 30 cm, corresponding to the zone where earthworms are expected to be most prevalent (Sims and Gerard, 1985). The excavated soil was spread out on a plastic sheet for hand-sorting and the total number of earthworms in each sub-plot was counted and recorded (Sims and Gerard, 1985).
2.2.4. Fecundity assessment To determine the fecundity of the earthworms, after counting the number in each segment at 7 d, the adult earthworms were removed and transferred to corresponding fresh treatments on other trays. The soil in the first was then seived through thress sieve sizes (3.2 mm and 6.7 mm) and the cocoons counted. The number of cocoons in the fresh treatments was determined after another 7 d and 14-d cumulative number of cocoons recorded.
2.2. Laboratory experiment 2.3. Data analysis The general experimental procedures were adapted from the OECD guideline (2015).
Data were first tested for normality and homogeneity of variance using the Kolmogorov-Smirnov and Levene’s tests, respectively. The tests showed that numbers of cocoons and live earthworms, and avoidance test data violated ANOVA assumptions even after transformation. Moreover, earthworm avoidance data for each concentration are dependent on counts in other concentrations, thereby violating the assumption of independence of observations. Accordingly, the nonparametric Kruskal-Wallis test was used to determine the effect of citrus waste concentration on number of cocoons and live earthworms, while Friedman’s test was used for avoidance data. In cases where significant differences were observed, post-hoc tests were conducted to separate means. Probit analysis was used to determine dose-response relationships (i.e. median effect concentration, EC50) between citrus waste concentration and earthworm response. Probit analysis has been used to determine dose-response relationships in earlier ecotoxocological studies using earthworms as a bioassay organism (e.g., Loureiro et al., 2005; Zhou et al., 2007). All statistical analysis were done using SPSS® version 16 (SPSS Inc., 2007).
2.2.1. Treatment preparations The laboratory experiment consisted of acute toxicity, avoidance and fecundity tests using Lumbricus terrestris L., positively identified at the Department of Biological Sciences at University of Zimbabwe. The choice of L. terrestris as a bioassay species was based on results of determination of field abundance indicating that this was the only species observed. Five concentrations of aqueous citrus extract solutions were prepared from fresh citrus Citrus sinensis L. waste (orange peels) collected from the waste dump site at MCE. The orange peels were crushed using a mortar and pestle. Aqueous citrus extract solutions of various concentrations were prepared by mixing known masses of the crushed fresh orange peels and volumes of deionized water. The following solutions, which constituted the treatments, were prepared on a weight/ volume (w/v) basis: 0% consisting of deionized water (control), 0.25:1 (25%), 0.5: 1 (50%), 0.75: 1 (75%) and 1: 1 (100% citrus extract). The solutions were left to stand for 2 d, after which they were filtered and used in the experiment.
3. Results
2.2.2. Acute toxicity test For the acute toxicity test, clean bulk soil samples, enough to fill four replicates of 1-L earthen pots (measuring 23 cm top diameter and 15 cm bottom diameter and 23 cm depth) per treatment were prepared and the five citrus extract solutions applied to the pots separately. The soil was thoroughly mixed with the citrus extract solutions, and then loosely packed in the 1-L pots. In each pot, ten mature, live earthworms (OECD, 2015) of length 5.5 ± 0.5 cm (mean ± standard error of the mean), were introduced in each pot. The earthworms were then covered with 5 cm of the corresponding treated soil. A dry mulch of couch grass, Cynodon dactylon (L.) Pers. cut by a lawn mower, was applied at
3.1. Soil chemical properties Analysis of selected soil chemical properties showed that the contaminated soil was acidic (4.8), while the uncontaminated soil was slightly alkaline (7.9) (Table 1). Citrus waste contaminated soil had significantly higher mineral N, available P and exchangeable Ca, Mg and K than the uncontaminated control. The shifts in chemical properties were about 1–2 orders of magnitude for P and Ca, two-fold for K and approximately 30–60% for pH, N and Mg. 3
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Table 1 Selected soil chemical properties for the citrus waste contaminated site and the uncontaminated control. Means with different letters are significantly different at probability p = 0.05.
Contaminated Uncontaminated
pH
Mineral N (mg/kg)
Available P, resin extract (mg/kg)
Potassium (mg/kg)
Calcium (mg/kg)
Magnesium (mg/kg)
4.8a 7.9b
52a 41b
151a 8b
11.0a 5.2b
407.4a 37.6b
55.3a 33.7b
Fig. 1. Mean number of live adult earthworms (L. terrestris) remaining out of the initial 10 per pot after the 7 and 14-d test periods (n = 4). Error bars denote standard error of the mean. Means for each concentration with different letters within each test period are significantly different at probability, p < 0.001.
3.2. Field earthworm abundance A t-test comparison of field counts showed that the citrus waste contaminated site had approximately 80% significantly (p < 0.001) lower earthworm abundance (0.8 ± 0.37 per m2) than the uncontaminated control (9.8 ± 0.81 per m2). In summary, results of both laboratory simulation and field measurements demonstrate that citrus waste extract had an adverse effect on earthworms. 3.3. Laboratory experiment 3.3.1. Acute toxicity effects on adult earthworms Citrus waste extract had significant (p < 0.05) biocidal effects on adult earthworms (Fig. 1). Significant (p < 0.001) reductions in the number of live adult earthworms were observed among all concentrations except 25% and 54%, which were similar. The number of live earthworms in the 100% citrus extract was about two times lower than that of the control. The number of live adult earthworms showed a strong exponential decline with increasing citrus extract concentration after 7 (y = 9.69e−0.006x, r2 = 0.96 d). A similar trend was observed after 14 d. However, for each citrus extract concentration, the numbers of live adults after 7- and 14-d exposure periods were similar.Probit analysis of data on live earthworms yielded mean effect concentration (EC50) of 83% citrus waste concentrations.
Fig. 2. Mean number of earthworms (L. terrestris) in compartments after a 2-d exposure to soils contaminated with various concentrations of citrus extract (n = 4). Error bars denote standard error of the mean. Bars with different letters are significantly different at p < 0.001.
observed in the avoidance test decreased exponentially with citrus waste concentration (y = 16.0 e−0.037x, r2 = 0.96, p < 0.001). The results clearly showed that avoidance was directly related to citrus extract concentration. The EC50 for earthworm avoidance behaviour was EC50 47% citrus waste concentrations.
3.3.2. Avoidance behaviour The number of earthworms in each compartment significantly (p < 0.05) declined with increasing concentration of citrus extract (Fig. 2). Earthworm counts varied significantly among all citrus extract concentrations, and declined significantly (p < 0.05) with increasing concentrations. Citrus waste-induced avoidance behaviour in earthworms changed by 44% to 100% compared to the control, with the earthworms completely avoiding the compartment with 100% citrus concentration. Compartments with 25% and 50% citrus extract concentrations accounted for 32% and 9% of the total number of earthworms introduced, respectively. The number of earthworm counts
3.3.3. Effects on fecundity The number of earthworm cocoons was used as an indicator of fecundity. Significant reductions (p < 0.002) in the number of earthworm cocoons were observed at different concentrations of citrus extract compared to the control (Fig. 3). An exception was the 50 and 75% concentrations which had similar numbers of cocoons. The number of earthworm cocoons (EC) exponentially declined with increasing concentration of citrus waste extract (C) (p < 0.001). The corresponding 4
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which include tannins, flavonoids and saponins which they showed to have biocidal effects on the root knot nematode, Melodogyne incognita. Davidson (2001) studied the effects of citrus extract on fleas and reported that D-limonene and linalool found in the citrus extract were responsible for the mortality observed on flea adults and larvae. D-limonene, an ingredient of orange oil has been demonstrated to be fatal against a variety of organisms which include ants, mosquitoes, flies, crickets and termites. The oil dissolves the insects’ exoskeleton, destroying the insect’s cell membranes. The loss of the exoskeleton in insects and other soil organisms consequently results in dying from excessive loss of water and proteins (Harris, 2013). The extraction and subsequent processing of citrus products also entail use of chemical additives. As shown by the data for the selected chemical properties, these chemical may accumulate in citrus waste and could have adverse impacts on chemical properties of soils. Therefore, besides the direct effects of chemical compounds originating from citrus, there is a possibility that these changes in habitat chemical properties induced by chemical additives may contribute to mortality and avoidance behaviour evident in both laboratory simulation and field studies. The increased avoidance behaviour of earthworms with increasing concentration of citrus extract observed could be ascribed to the strong olfactory senses that the organisms use to identify food and to avoid poisonous environments (Tsai, 2008). This implies that as the concentration of the citrus extract increased, the odour of the active compounds also increased and the earthworms avoided them. Iordache et al. (2010) also carried out avoidance tests on earthworms using inorganic fertilisers and obtained almost similar results. They attributed such behaviour to the earthworms’ olfactory capabilities. Therefore our results suggest that earthworms preferentially avoid soils contaminated with citrus waste. Citrus contamination also had significant negative effects on the fecundity of earthworms. It reduced the number of cocoons produced by earthworms, probably also contributing to low earthworm populations in the soil. This implies that soils contaminated with citrus waste are not a conducive habitat for earthworms. Kirkpatric (2003) studied optimum conditions necessary for earthworm survival and observed that under ‘stressful’ conditions, earthworms lose their clitellum (glandular swelling on earthworms). The clitellum is involved in reproduction (Donahue, 2003). This is the most probable cause in the decrease in fecundity and also the decrease in population caused by the citrus extract. The low earthworm population in the contaminated plots could also be ascribed to the fact that if earthworms have to “fight” against the limiting factors such as the contaminants or the low pH, they use their energy reserves for that, instead of the biological processes such as reproduction and growth according to the theory of dynamic energy budget (Kooijman, 2009). Butt (1991) showed that with food supply L. terrestris, fecundity decreased from 1.9 cocoon per month at 18–21 °C to less than 1 cocoon per month as temperature rose to 22 °C. Mean ambient temperatures during the current study were more than 25 °C which would negatively affect the earthworms. The drop in pH from 7.9 for the untreated control to 4.8 for the citrus waste contaminated soils was attributed to organic acids (e.g. citric and limoninic acids) derived from citrus wastes (Kimball, 1999). The acidic pH was below the near-neutral (6.5–7.5) considered optimum for earthworms (Edwards and Bohlen, 1996), and could partly account for the reduced fecundity and survival observed in citrus waste contaminated soils. The profound adverse effects on soil pH could be attributed to the very high citrus waste application rates (i.e., 15 cmlayer equivalent to 10000 Mt/ha/year). Assuming the acidic soil pH is one of the key factors accounting for the adverse impacts of citrus waste on soil biota, two options exist to minimize the ecological impacts. First, future disposal should reduce annual application rates from the current 15 cm or 10000 Mt/ha/year by about one to three orders of magnitude to attain typical maximum recommended rates (150–250 Mt/ha/year) for bio-solid land application (USEPA, 2000).
Fig. 3. Mean number of earthworm (L. terrestris) cocoons after 7- and 14-d exposure periods (n = 4) for an initial 10 earthworms per pot. Error bars denote standard error of the mean. Means with different letters are significantly different at probability, p < 0.001. Note that cocoons were recorded and discarded after the first 7 d and thereafter earthworms placed back for another 7 d to make a cumulative 14 d.
exponential decay functions for the 7- and 14-d exposure periods were: EC7-d = 63.1e0.49C (r2 = 0.98) and EC14-d = 70.5e0.43C (r2 = 0.99), respectively. Across the various citrus extract concentrations, the number of cocoons for the 7-d exposure period was generally higher than that for the 14-d period. The EC50 for numbers of cocoon was approximately 91% citrus waste concentrations.
4. Discussion The results show that the citrus waste has significant effects on earthworm abundance in soils. Concentrations as low as 25% showed mortality rates of at least 20% and almost 50% mortality at concentrations around 75%. The longer the exposure period, the higher the mortality with mortality increasing by almost 90% at the highest level of contamination with citrus waste after 14 d. Sampling at the Mazowe dumpsite showed that citrus waste has severe effects on the populations of earthworms, with populations at the uncontaminated sites at least six times greater than those at the contaminated site. The general low numbers of earthworms record at both sites could attributed to the fact that the study was conducted towards the end of the wet season. Gelsomino et al. (2010) highlighted that the alternative use of citrus processing waste as soil conditioner in agriculture has been investigated without encouraging results. They pointed out that repeated application of large amounts of citrus waste have shown evidence of significant limitations. Citrus waste application to agricultural soils has shown notable negative effects on both soil chemical properties and crop yields (Bellingo et al., 2005). Loss of soil quality due to elevated metal ions leads to decreased yields due to decreased pH, loss of basic cations, inhibition of nitrification, promotion of certain diseases and fixation of major anion nutrients (Gupta, 2011). Some of these adverse effects of citrus waste result in decrease soil faunal population. Survival and reproduction of earthworms decreased with increasing concentration of citrus waste extract. Avoidance also increased with increase in concentration; with no earthworms found in the portion with 100% citrus extract concentration. Lower EC50 (47%) was observed for avoidance behaviour than numbers of cocoons (91%) and live earthworms (83%), suggesting that earthworm avoidance behaviour was more sensitive to citrus waste concentration than fecundity and survival. The effects on earthworm survival were probably due to the citrus waste containing toxic compounds that have serious biocidal effects on earthworms. It was not the objective of this study to find the chemical composition of the citrus waste but previous studies by Abolusoro et al. (2010) suggested that citrus peels contain bioactive organic compounds 5
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sequestration and nutrient availability after different organic matter inputs in virgin and cultivated Zimbabwean soils. Commun. Soil Sci. Plant Anal. 32, 877–894. Asgher, M., Bhatti, H.N., 2012. Evaluation of thermodynamics and effect of chemical treatments on sorption potential of citrus waste biomass for removal of anionic dyes from aqueous solutions. Ecol. Eng. 38, 79–85. Bellingo, A., Di Leo, M.G., Marchese, M., Tuttobene, R., 2005. Effects of industrial orange waste on soil characteristics and on growth and production of durum wheat. Agron. Sustain. Dev. 25, 129–135. Blouina, M., Hodsonb, M.E., Delgadoc, E.A., Bakerd, G., Brussaarde, L., Buttf, K.R., Daig, J., Dendoovenh, L., Peresi, G., Tondohj, J.E., Cluzeauk, D., Brunl, J., 2013. A review of earthworm impact on soil function and ecosystem services. Eur. J. Soil Sci. 64, 161–182. Butt, K.R., 1991. The effects of temperature on the intensive production of Lumbricus terrestris L. (Oligochaeta: Lumbricidae). Pedobiologia 35, 257–264. Chauhan, R.P., 2014. Role of earthworms in soil fertility and factors affecting their population dynamics: a review. Int. J. Res. 1, 642–649. Chubukov, V., Mingardon, F., Schackwitz, W., Baidoo, E.E., Alonso-Gutierrez, J., Hu, Q., Lee, T.S., Keasling, J.D., Mukhopadhyay, A., 2015. Acute limonene toxicity in Escherichia coli is caused by limonene hydroperoxide and alleviated by a point mutation in alkyl hydroperoxidase AhpC. Appl. Environ. Microbiol. 81 (14), 4690–4696. Davidson, N.A., 2001. Least-toxic Alternatives for Argentine Ants, Fleas and White Grubs of Lawns. Department of Pesticide Regulation, California, USA. Donahue, W.A., 2003. Contact Pesticidal Effects of D-Limonene on Argentine Ants. John Wiley and Sons, New York, USA. Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms, 3rd edition. Chapman and Hall, London. Garcia, M., Rombke, J., de Brito, M.T., Scheffczyk, A., 2008. 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This can be achieved by spreading the citrus waste over a larger area. Second, soil acidity on existing citrus waste contaminated soils can be neutralized through application of lime and/or other readily available low-cost alkaline amendments such as coal ash and ash from combustion of solid waste. In this regard, application of lime and/or other alkaline amendments could aim to achieve a pH of 6.5, which could in turn, promote long-term ecological recovery. Although the current study used earthworms as a bioassay organism, the results may have implications on other soil macro-invertebrates and possibly other soil organisms. The observed mortality and avoidance behaviour of the earthworm imply that citrus wastes may undergo slow decomposition process due to their potentially toxic effects on soil organisms. This has implications on the fate of such wastes when disposed of in waste repositories such as landfills, land application and their use in waste-to-energy recycling programs. For instance, in biogas digesters, putative changes in digestate pH and microbial communities caused by use of citrus wastes may adversely affect biogas production. Indeed, several studies investigating biogas production from citrus waste confirm that the presence of toxic compounds such as limonene adversely affect the use of such wastes as biogas feedstock if no pre-treatment is done (Martin et al., 2010; Nguyen, 2012; Wikandari et al., 2014). 5. Conclusion Citrus solid waste had adverse effects on the survival, fecundity, and avoidance behaviour of earthworms. Fecundity, survival and number of earthworms in avoidance test declined exponentially with increasing citrus waste concentration. However, mean effect concentration (EC50) showed that earthworm avoidance behaviour was more sensitive to citrus waste concentrations than fecundity and survival. These adverse could be attributed to acidic pH and potentially toxic organic compounds in citrus waste such as limonene. Our results demonstrate that soil biota can be negatively affected by excessive exposure to citrus processing waste, pointing to the need for caution when disposing such wastes on land. Long-term solutions may include proper citrus processing waste disposal practices such as engineered landfills and conversion to value-added products such as stockfeed are required to prevent the adverse ecological impacts. Possible options to minimize the ecological impacts of citrus solid waste include: (1) reducing current application rates to maximum internationally recommended rates for land application of bio-solids by spreading the citrus waste over a large area; and (2) neutralizing soil acidity through application of lime and/or other readily available low-cost alkaline materials such as coal ash and ash from combustion of solid waste. Further research on already contaminated sites could investigate phytoremediation with acid-tolerant plant species such as vetiver grass and star grass. Acknowledgements We are grateful to the Mazowe Citrus Estate (MCE) for allowing us to conduct this study on their premises. We acknowledge technical and logistical support provided by the staff in the Department of Soil Science and Agricultural Engineering, University of Zimbabwe (UZ). The expertise provided by staff in the Department of Biological Sciences, UZ in correctly identifying the earthworm species is greatly appreciated. References Abolusoro, S.A., Oyedunmade, E.A., Olabiyi, T.I., 2010. Evaluation of sweet orange peel aqueous extract (Citrus sinensis) as root-knot nematode suppressant. Agro-Sci. J. Trop. Agric. Food Environ. Ext. 9, 170–175. Alburquerque, J.A., de la Fuente, C., Bernal, M.P., 2012. Chemical properties of anaerobic digestates affecting C and N dynamics in amended soils. Agric. Ecosyst. Environ. 160, 15–22. Almendros, G., Giampaolo, S., Pardo, M.T., 2001. Laboratory appraisal of carbon
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