The utilization of poultry manure as an organic fertilizer is essential in improving soil productivity and crop production (Dikinya and Mufwanzala 2010). While the use of organic wastes as manure has been in practice for centuries world-wide and in the recent times (López-Masquera et al. 2008), there still exists a need to assess the potential impacts of poultry manure on soil chemical properties and leachates and in particular evaluating the critical application levels (Delgado et al. 2010).

The MS3, from two types of poultry manure (sawdust and straw bed), has also proved to be functional for assessing effects on earthworm, plants and microorganisms on an agricultural land (Delgado et al. 2012), and combined pollutants in contaminated sites (Fernández et al. 2005).

This paper reports the effects on leachates organisms (aquatic invertebrates, algae and in vitro cell line on fish), after applying two types of poultry manure (sawdust or straw bed) on an agricultural land.

Materials and Methods

Multi-species-soil-system test (MS3) is a well-known tool used in order to develop a toxicity test soil and poultry manure (Delgado et al. 2012).

The MS3 was saturated (spring water; 730–940 mL/MS3). After 14 days, no leachates were produced yet and it was decided to keep the irrigation for two more days, until saturation (450 mL, spring water), in order to obtain an acceptable amount of leachates. Chemical properties and ecotoxicity test (Daphnia magna, Chlorella vulgaris and in vitro toxicity on RTG-2 cell) were carried out on the leachates.

Crustacean (D. magna) acute immobilisation test was conducted following the methodology described in ISO guidelines (ISO 1996) with some modifications. For all the samples range finding test were carried out prior to EC50 determinations (OECD 2004). In order to study the acute toxicity test on D. magna the trials were conducted in reconstituted water to Daphnia, for 24 h, after having been kept in agitation with poultry manure. Tests were made at the following dilutions of this extract: 50 %–25 %–12.5 %–6.25 %–3.12 % and 1.56 %. At 48 h there were dead and living individuals. The DL50 (dilution causing 50 % mortality) and its confidence limits 95 % were calculated using the analysis program probit version 1.4. The regression model was verified using Chi square test and T-Student ANOVA to determine the presence of significant differences between the control and treatment groups. These results are incompatible with those obtained by Slomczynska and Slomczynski (2004) using the D. magna test: In this case the period of 24 h was too short to allow the manifestation of the toxic effect.

Algae (C. vulgaris), is a planktonic unicellular green alga growth inhibition test (method described in Ramos et al. 1996). Algae growth was estimated by measuring increases of absorbance (λemission = 450 nm), fluorescence (indicating variations in the chlorophyll content, λemission = 680 nm, λexcitation = 430 nm) and by direct counting using a Neubauer chamber (OECD test guideline 202).

The rainbow trout fish cell line RTG-2 (ATCC CCL55) was grown at 20°C in an incubator and propagated in eagle’s minimum essential medium (EMEM) supplemented with 10 % foetal bovine serum, l-glutamine, non-essential amino acids and penicillin–streptomycin. The cytotoxicity assay was conducted according to Babin and Tarazona (2005). Cell viability was quantified using the neutral red (NR) uptake assay (Borenfreund and Puerner 1985). Presence of co-planar organic toxic chemical was indicated by the stimulation of cytochrome CYP1A, which was measured as 7-ethoxyresorufin-O-deethylase (EROD) activity (Babin and Tarazona 2005). Cellular defence was predicted by hydrolysis of 4-methylumbellferyl-β-D-galactoside (β-gal activity) assay (Babin and Tarazona 2005). The TECAN-Genius spectroflurometer was used to quantify fluorescence (kinetic way) and absorbance endpoints.

Statistical differences for chemical properties of leachates, between poultry-manure amended soil treatments and the control, were assessed by analysing the variance (p ≤ 0.05 and p ≤ 0.01), one-way ANOVA test and the least significant difference (LSD) multiple range test at p ≤ 0.05 and p ≤ 0.01 (SAS Institute 2001).

Results and Discussion

Table 1 reflects the analytical characterization of leachates (volume, pH, electrical conductivity and nitrate). It can be observed a high nitrate concentration in the leachates from the sawdust and straw poultry manure, at time 0 day and 12 days.

Table 1 Volume, pH, electrical conductivity and nitrate (mean ± SD)
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On D. magna was high mortality for zero time and almost 100 % and 70 %–80 % mortality for sawdust and straw poultry manure respectively. It can be due the high nitrate concentration in the leachates from both poultries manure.

Figures 1 and 2 show the graphics of the adjusted models (confidence limits 95 %), for the variables: ‘effects’ versus ‘the percentages of dilution for sawdust and straw poultry manure’ respectively.

Fig. 1
figure 1

Acuate Dapnia test, Probit analysis for LD50 of straw poultry manure

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Fig. 2
figure 2

Acuate Dapnia test, Probit analysis for LD50 of sawdust poultry manure

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The percentiles of the adjusted models indicate the value of dilution, in percentage, at which the model reaches certain values. The DL50 had percentile 15.77 (13.43–18.85 95 % CI) for sawdust poultry and percentile 11.06 (9.61–13.13 95 % CI) for straw poultry manure.

The effects on C. vulgaris and in vitro toxicity on RTG-2 cells exposed to leachate were:

No effects, on the inhibition growth of C. vulgaris, were observed after the exposure period (72 h) at any leachates from two different poultry manure. Similar effect was detected by Navas et al. (2006) in the water soluble fraction of the fuel oil.

The cytotoxicity assessment of leachates did not reveal toxicity on the poultry manure amended soil. Neutral Red and protein level estimations, as indicators of cytotoxicity indexes, were similar in all treatments (Figs. 3, 4 respectively).

Fig. 3
figure 3

Neutral Red (absorbance) on RTG-2 fish cell lines

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Fig. 4
figure 4

Proteins (absorbance) on RTG-2 fish cell lines

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None of the parameters considered; β-gal activity (Fig. 5) and EROD activity (Fig. 6), showed differences between the control and the leachate collected after exposure to poultry manure.

Fig. 5
figure 5

β-Gal activity (fluorescence) on RTG-2 fish cell lines

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Fig. 6
figure 6

EROD activity (fluorescence) on RTG-2 fish cell lines

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Finally, the capacity of the in vitro assays on fish cell lines, as a complementary tool for analysing effluents, wastes and hot spots has been previously demonstrated (Vega et al. 1996) but limitations should also be considered.