Agronomic Assessment of Olive Mill Wastewater Sludge Derived Composts on Lactuca sativa and Zea mays: Fertilizing Efficiency and Potential Toxic Effect on Seed Germination and Seedling Growth

Abstract

Olive mill wastewater is a polluting residue generated from the olive oil industry and is one which constitutes an environmental concern in Mediterranean countries. Composting has been reported as a viable valorization alternative, as it reduces the volume and the phytotoxic characteristics of OMW. In this study, several composts derived from OMW were evaluated under controlled conditions over two growing season pot experiments using Lactuca sativa as a test crop. The analysis focused on soil quality changes, crop yield, and plant development. Additionally, potential phytotoxicity was also evaluated through a direct acute toxicity plant growth test. Application of OMW composts improved soil fertility indicators, including oxidizable carbon, Kjeldahl total nitrogen, Olsen phosphorous, and plant availability. Crop yields were comparable to those obtained with other organic amendments such as vermicompost and fresh cattle manure in both growing seasons and plant development (in terms of chlorophyll content and canopy cover) was not negatively affected. Nutrient uptake (NPK) was consistent during both growing seasons, with similar nitrogen use efficiency to that achieved in other organic treatments. Regarding the potential toxic effect, the OMW composts tested enhanced seed germination when mixed with coconut fiber at weight ratios below 29.2%. No half-maximal effective concentration (EC50) values were detected, even at 100% compost concentration, while half-maximal inhibitory concentration (LC50) values ranged between 65–75%. These results indicate that OMW composts can serve as an effective short-term source of plant-available nitrogen and a medium-term source of phosphorus, without risk of finding inhibitory or phytotoxic effects on crops.

1. Introduction

One of the main environmental concerns from the olive oil industries is the disposal of olive oil mill wastewater (OMW). Olive oil in Mediterranean countries accounts for nearly 90% of worldwide production [1], which implies that waste derived from this agro-industry is highly concentrated in some regions [2]. OMW presents significant management challenges due to its physicochemical characteristics. OMW is rich in organic matter (OM) and phytotoxic compounds, such as phenolic compounds with a strong odor, and can cause severe soil and water pollution, inhibiting plant growth, exacerbating soil hydrophobicity and disrupting ecosystemic balance [3,4,5,6].

Traditionally, OMW has been managed through evaporation in drying ponds. However, this method has proven inadequate for handling this hazardous waste, mainly due to poor maintenance of the ponds and the subsequently high environmental impact [7] and the recalcitrant characteristics of OMW [8]. Through different techniques, OMW has proven to have great potential for the obtention of different high-value products such as organic amendments, compounds of pharmaceutical interest, or bio-stimulants for agricultural purposes [9]. Other strategies have been addressed, such as ultrafiltration/reverse osmosis [10,11], flocculation clarification [12], thermal concentration and evaporation [13], incineration and combustion, and gasification [14,15]. However, these strategies are costly, present a high technical requirement, and the pollution problems are not sufficiently reduced. Therefore, more research about viable technological procedures to manage these wastes is needed. In this context, some authors have proposed biological methods, such as composting, as the most cost-efficient management systems [16,17].

As the use of inorganic fertilizers in the EU rises, it creates dependence on external industries, which in turn rely on high carbon footprint processes like the Haber–Bosch process [18,19]. However, these fertilizers tend not to improve soil health nor to replace organic matter [20]. Thus, N- and K-rich fertilizers of inorganic sources are used to compensate for nutrient-poor soils [21].

Optimizing nutrient application is key to reducing negative effects on the environment and increasing the sustainability of ecosystems. Applied fertilizers though several consecutive crop seasons either accumulate in the soil or are lost by lixiviation or runoff when the N supply exceeds the demand of the crops, causing several physiological responses that can reduce the profit of growers [22]. As the nutrient content of composts heavily depend on the initial materials, OMW composts can inherit some of their characteristics, like phytotoxicity or low nutrient availability. Biochar, a product derived from the pyrolysis of biomass and which can be used as a soil amendment, can enhance nutrient availability in some agricultural soils when applied with compost [23]. Thus, testing OMW composting systems with Biochar addition can result in improved agronomic characteristics and lower environmental impact.

This study aims to investigate the feasibility of composting to reduce the toxic effects and limiting characteristics commonly associated with OMW, enabling its use as an agronomic fertilizer. For this purpose, different OMW-derived composts were tested in an experiment with lettuce (Lactuca sativa) under controlled conditions, in order to assess the viability of the composts as soil amendment, their effect on crop production and development, and the impact of their application on soil during two growing seasons. The potential acute toxicity of composts was also evaluated through an experiment on maize (Zea mays), using different concentrations of composts blended with coconut fiber to determine their half-maximal inhibitory concentration (LC50) and half-maximal effective concentration (EC50).

2. Materials and Methods

2.1. Pot Experimental Design

A two-growing season pot experiment with lettuce (Lactuca sativa L. “Celistra”) was carried out in the FertiLab-EPSO UMH (Spain) under controlled conditions. During both growing seasons, the temperature was between 21 and 25 °C, the relative humidity was 50–60%, and the photoperiod was 12 h/12 h (light/dark, respectively) through artificial lamps (RX600, Solray^® 285, Helsinki, Finland). Plastic pots (Ø 11 cm, 1200 cm³) were filled with 1500 g of soil, prepared according to the OECD 207. A total of 1984 soil-based plant tests were undertaken, consisting of a mixture of natural, loamy soil (0–20 cm depth, air-dried, and 5 mm sieved) collected at the EPSO-UMH (38°4′9.066″ N, 0°59′6.148″ W mixed with fine, and coarse sand (50:25:25% w:w:w, respectively). The resulting synthetic soil presented a granulometric distribution of 60% sand, 12.5% silt, and 27.5% clay, classified as a sandy loam texture (USDA taxonomy) with a bulk density of 1.27 kg dm⁻³, a pH of 8.47, and an electrical conductivity (EC) of 3.58 mS cm⁻¹.

The fertilization treatments were superficially applied according to a normalized N application rate of 200 kg N ha⁻¹ (except IN100, which was adjusted to 100 kg N ha⁻¹). A total of 8 composts were tested, all with OMW as their main ingredient (

Table 1. Fertilizer treatments applied to the pot test for evaluation.

Type	Acronym	N Source	C Source	Biochar	Treatment
Reference	Control	-	-	-	No fertilizer treatment applied
Complex (15–15–15)	IN100	-	-	-	Inorganic fertilizer (15–15–15) rate 100 kg N/ha
Complex (15–15–15)	IN200	-	-	-	Inorganic fertilizer (15–15–15) rate 200 kg N/ha
Vermicompost	Vermi	-	-	-	Vermicompost
Fresh manure	Manure	-	-	-	Fresh CM in direct application
Compost	T1-C	CM	Alm	No	OMW 50% + CM 40% + Alm 10%
Compost	T1-B	CM	Alm	Yes	OMW 50% + CM 40% + Alm 10% + 1% f.w. Biochar
Compost	T2-C	CM	Vn	No	OMW 50% + CM 40% + Vn 10%
Compost	T2-B	CM	Vn	Yes	OMW 50% + CM 40% + Vn 10% + 1% f.w. Biochar
Compost	T3-C	GM	Alm	No	OMW 50% + GM 40% + Alm 10%
Compost	T3-B	GM	Alm	Yes	OMW 50% + GM 40% + Alm 10% + 1% f.w. Biochar
Compost	T4-C	GM	Vn	No	OMW 50% + GM 40% + Vn 10%
Compost	T4-B	GM	Vn	Yes	OMW 50% + GM 40% + Vn 10% + 1% f.w. Biochar

OMW: olive mill wastewater sludge; CM: cattle manure; GM: goat manure; Alm: almond tree pruning; Vn: vineyard pruning waste; f.w.: fresh weight.

). As positive controls, three different treatments were applied—inorganic fertilizers with NPK content of 15–15–15%, vermicompost produced using goat manure, and fresh cattle manure. Additionally, three pots did not receive any fertilizer treatment and were used as blank. One seedling of Lactuca sativa L. “Celistra” was sown in each pot. All treatments were applied in triplicate (n = 3) and randomized placed and irrigated to constantly maintain the soil at 60% of its water-holding capacity. Applied treatments are displayed in Table 1.

The growing season lasted for 51 days until harvest. After harvesting the lettuce, the soil from each pot of the same treatment was combined for its use for analytical purposes and was reused as a substrate for a second growing season. The second growing season lasted for 48 days until harvest under the same conditions as the first growing season, including lettuce variety and environmental conditions. No fertilizer treatment was applied to the soil before the second growing season. The harvesting was carried out as per the first growing season and the soil was used for analysis.

2.2. Compost Production

All composts used for this test were produced during the experiments developed by Mira-Urios et al. [24]. The composting process was carried out in the CompoLab treatment plant of the EPSO (Orihuela, Alicante, Spain) in a windrow composting system. All piles were turned 4 times and the entire process consisted of 119 days of the bio-oxidative phase and 46 days of the maturation phase, for a total of 165 days. The materials utilized were olive mill wastewater (OMW) sludge as main ingredient (50% f.w.) in all piles, cattle manure (CM) (T1 and T2) or goat manure (GM) (T3 and T4) as the N source, and almond tree pruning (Alm) or vineyard pruning waste (Vn) as the C source and bulking agent. In addition, in other replicates of each mixture, Biochar obtained from the pyrolysis process of urban pruning waste (450 °C, 24 h) was applied as an additive (1% f.w.). The physicochemical characteristics of the resulting composts were analyzed following the methods of [25] and are shown in

Table 2. Physico-chemical characteristics of the compost treatments.

Compost	Vermi	Manure	T1-C	T1-B	T2-C	T2-B	T3-C	T3-B	T4-C	T4-B
pH	7.48	8.80	8.72	8.39	8.60	8.57	9.62	9.30	9.00	8.84
EC (dS/m)	1.74	8.52	9.61	10.3	9.19	9.23	8.41	9.48	7.84	7.65
TOC (%)	32.0	42.6	36.7	64.6	32.7	58.7	60.0	91.3	54.4	55.0
TN (%)	2.88	3.05	3.04	3.13	2.79	2.79	2.63	2.58	2.49	2.48
TOC/TN	11.4	14.0	12.1	11.5	11.7	11.7	12.4	13.1	30.5	12.4
PPH (mg/kg)	250	-	3649	3679	3913	4190	4551	5558	3212	2763
Total elements
P (g/kg)	1.00	1.92	0.98	1.28	0.89	0.89	1.45	1.29	1.04	1.10
K (g/kg)	1.94	1.65	3.02	3.19	3.32	3.42	3.75	3.89	3.23	3.09
Ca (g/kg)	7.81	2.23	3.81	4.12	4.42	4.38	4.22	3.82	4.92	4.86
Cd (mg/kg)	<0.01	0.14	0.28	0.33	0.30	0.34	0.30	0.36	0.31	0.32
Cr (mg/kg)	13.2	7.91	22.2	26.0	32.5	29.0	22.4	27.21	36.8	36.5
Cu (mg/kg)	60.9	33.1	70.3	72.9	67.5	68.3	58.2	54.5	59.5	60.0
Ni (mg/kg)	7.13	6.04	0.67	0.71	0.67	0.69	0.80	0.82	0.61	0.57
Pb (mg/kg)	13.1	0.93	9.10	10.44	11.6	10.6	10.3	11.2	12.7	12.6
Zn (mg/kg)	1046	130	6.69	7.20	12.2	9.11	6.87	6.07	9.36	9.52

EC: electrical conductivity; TOC: total organic carbon; TN: total nitrogen; PPH: water-soluble polyphenols.

.

2.3. Test of Phytotoxicity

An indirect germination index (GI) test was performed for all composts using water extracts (1:10 w/v) and seed of Lepidium sativum L. in accordance with the method described by Zucconi et al. [26]. The GI was calculated from the percentage of seed germination (G) and root elongation (R), determined by comparison with the results of the control using the following Formula (1):

$$GI={\displaystyle \frac{[G\times R]}{100}}$$

(1)

The two composts that obtained the highest germination index (GI) were selected (T1 and T4) and the potential phytotoxicity of the composts was evaluated through a direct acute toxicity plant growth test (OECD 208), using different proportions of compost mixed with coconut fiber (100/0, 50/50, 25/75, 12.5/87.5 w/w %). Around 60 g of substrate was placed in plastic seedbeds, with 10 replicates for each mixture and one seed of maize (Zea mays L. var. “Rostrato”). The plants were left for 2 weeks to grow after 50% of seeds in the control treatment had emerged. Humidity of the substrates was maintained at 70% of their water-holding capacity. The number of emerged plants in each treatment was recorded and the fresh and dried weight of their aerial parts and roots were determined. The Half maximal effective concentration (EC50) and median lethal concentration (LC50) were calculated by applying a linear regression analysis to the relationship between the logarithm of the percentage compost concentration and the toxic effect percentage on plant growth (fresh and dry weight) and seedling emergence with respect to the control (100% coconut fiber without compost) [27].

2.4. Sampling and Analytical Methods

2.4.1. Soil Analysis

After each growing season, all the soil from pots with the same treatment were integrated and used as samples for analysis. This soil samples were air-dried after removal of all large roots, ground and sieved to <2 mm. Samples were analyzed for pH in a 1:2.5 soil/water extraction (w/v) and EC in a 1:5 soil/water extraction (w/v), while total Kjeldahl nitrogen (TKN) [28], Olsen phosphorus [29], plant-available potassium (Kava) [30], and oxidizable carbon (Cox) were determined by a modified Walkley and Black method [31].

2.4.2. Plant Development and Vegetal Tissue Analysis

In both growing seasons, the green cover factor (fCOVER) and chlorophyll content (CCC) were measured for each plant after 10, 25 and 45 days. fCOVER was determined using Canopeo [32] and CCC was determined using a hand-held chlorophyllmeter (Konica Minolta Chlorophyll Meter SPAD-502Plus) [33].

The first growing season harvesting was performed after 51 days of the experiment and the second growing season harvest was carried out 48 days after the second planting. Fresh aboveground biomass was weighted (g pot⁻¹), then dried at 60 °C for 48 h, and lastly dry biomass was weighted (g pot⁻¹). Dry samples were ground and sieved (<1 mm) for chemical analysis. Total C (TOC) and N (TN) content was analyzed using an automatic elemental microanalyzer (EuroVector, Milan, Italy). Macro and micronutrients (Ca, S, Mg, Cu, Fe, Mn, Zn, Na, P and K) were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) after a HNO₃/HClO₄ (1:4 v/v) digestion.

Nutrient efficiency of the first growing season for N (NUE) was calculated as the ratio between the nutrient application rate from the treatments and the nutrient uptake [34].

2.4.3. Statistical Analysis

Statistically significant differences between the treatments were assessed by one-way analysis of variance (one-way ANOVA), with each treatment as a factor, using the IBM SPSS Statistics 22.0 software. The normality of the data was tested using the Shapiro–Wilks test, and the homogeneity of variance was assessed using the Levene test (p > 0.05). The Tukey b test was used to analyze significant differences and multiple comparisons between the different treatments at a 5% significance level (p < 0.05).

3. Results and Discussion

3.1. Soil Parameters

The soils where compost treatments were applied showed differences in Cox at the end of the first growing season, with values higher than inorganic treatments and similar to the other organic treatments—manure and vermicompost. After the second growing season, Cox values of the soils with compost were similar to or higher than the other organic treatments. The only exception to the general behavior was T4-B, which showed the highest values of Cox after the first growing season and the lowest after the second growing season. The increase in the Cox after compost application shows an increment of the C stock of the soil and an increase in the edaphic organic matter [35,36]. Other studies have also reported that OMW, provided that their phytotoxic effects are neutralized, can be used as soil amendments in semiarid areas poor in organic matter [37]. de Sosa et al. [38] reported a cumulative effect in C sequestration in soil treated with urban and agro-industrial compost in an olive crop under Mediterranean conditions during four consecutive years.

The application of compost treatments caused an increment in KTN of the soils at the end of the first growing season with overall values similar to inorganic treatments. Nonetheless, soils with T1 treatments had higher KTN than inorganics. All fertilizer treatments had statistically higher values than the control treatment without fertilization, with the treatment with vermicompost being the highest. Doublet et al., [35] found that the 0–50 µm fraction particle size presented the most humified organic matter and contributed the most to the N mineralization of sludge and N availability after compost application in soil.

Regarding Olsen-P, at the end of the first growth session, and as expected, the statistically highest values were determined in the IN200 treatment. This treatment had a phosphorus application ratio per pot 2.2–2.5 times higher than the compost treatments. In this sense, OMW composts, such as T1-C, T2-B and T3-B, presented similar final values than IN100 treatment. At the end of the second growing season, the same trend was observed but with smaller differences, even without statistical differences when comparing the IN200 treatment with T1-B, T1-C, T2-C, T3-C and T3-B treatments. The soil of treatment T2-B presented the highest Olsen-P content. Grigatti et al. [39], in a pot test with ryegrass, reported that bio-waste digestate, and sewage sludge compost in particular, ensures P availability for two growth cycles due to their proportions in P forms, with 26–38% of P-labile and 5–11% of extractable-P (NaOH-P), which are recognized to be available immediately for plants and over the medium- and long-terms, respectively [40]. It appears, therefore, that the relative efficiency in P supply of the OMW compost tested could be similar to this type of compost.

At the end of the first growing season, composts with OMW compost treatment showed significantly higher Kava than soils with any other treatment. This behavior was demonstrated in particular in the T3 and T4 composts (with GM), which exceeded inorganic treatments. T1 and T2 showed this effect but not to the extent of the previous treatments and on some occasions the Kava was found to be similar to inorganic treatments. After the second growing season, all OMW composts showed this increment in Kava over inorganic treatments and, in some cases, similar to vermicompost and manure (

Table 3. Soil parameters at the end of each growing season depending on the application of different treatments.

Treatment	pH	EC (mS/cm)	Cox (g/kg)	KTN (g/kg)	Olsen-P (mg/kg)	Kava (mg/kg)
First Growing Season
Control	7.96 c	2.70 b	3.47 ab	0.40 a	21.9 a	276 a
IN100	7.89 abc	2.71 b	3.40 a	0.51 ab	32.4 c	254 a
IN200	7.86 abc	2.70 b	3.40 a	0.51 ab	45.5 d	389 bcd
Vermicompost	7.90 abc	2.74 b	4.47 de	0.70 d	25.8 ab	350 b
Manure	7.83 abc	2.26 a	4.17 cde	0.50 ab	21.9 a	375 bc
T1-C	7.85 abc	2.73 b	3.97 bcd	0.68 cd	32.7 c	406 bcd
T1-B	7.91 bc	2.67 b	3.88 abc	0.65 cd	30.8 bc	441 de
T2-C	7.70 ab	2.78 b	3.95 bcd	0.50 bc	30.9 bc	433 cde
T2-B	7.68 ab	2.68 b	4.38 cde	0.62 bcd	33.2 c	465 ef
T3-C	7.75 abc	2.77 b	4.23 cde	0.50 bc	27.7 bc	567 g
T3-B	7.69 ab	2.70 b	4.03 cd	0.55 bc	32.8 c	516 f
T4-C	7.77 abc	2.63 b	4.27 cde	0.48 ab	27.5 b	507 f
T4-B	7.67 a	2.61 b	4.63 e	0.58 bcd	26.6 ab	516 f
F-Anova	4.55 ***	5.57 ***	10.3 ***	7.83 ***	18.2 ***	49.3 ***
Second Growing Season
Control	7.50 ab	1.89 ab	3.47 ab	-	20.5 ab	143 a
IN100	7.59 ab	1.90 abc	3.97 abc	-	28.5 bcd	207 bc
IN200	7.62 ab	1.87 a	4.03 abc	-	31.1 d	183 ab
Vermicompost	7.63 ab	1.88 ab	3.43 a	-	23.2 abc	242 cd
Manure	7.66 ab	1.95 abc	3.60 ab	-	23.2 abc	232 bcd
T1-C	7.46 ab	1.87 a	4.55 c	-	29.6 cd	249 cd
T1-B	7.38 a	2.08 abc	4.22 abc	-	28.6 bcd	242 cd
T2-C	7.57 ab	2.10 cd	4.33 bc	-	29.5 bcd	247 cd
T2-B	7.48 ab	2.06 abc	3.83 abc	-	32.6 d	227 d
T3-C	7.60 ab	2.13 d	3.82 abc	-	27.1 bcd	285 d
T3-B	7.51 ab	2.02 abc	4.03 abc	-	27.1 bcd	297 d
T4-C	7.75 b	2.12 d	3.72 abc	-	21.0 a	254 cd
T4-B	7.63 ab	2.06 abc	3.37 a	-	22.6 abc	267 d
F-Anova	2.98 **	5.20 ***	4.65 ***		7.16 ***	11.1 ***

EC: electrical conductivity; Cox: oxidizable carbon; KTN: Kjeldahl total nitrogen; Olsen-P: Olsen phosphorous; Kava: available potassium. **, ***: significant difference between treatments at p < 0.01, and p < 0.001, respectively; ns: not significant. Different letters within a column indicate significant differences between treatments (p < 0.05).

). Piotrowska et al. [41] also reported increments of Kava in soils after applying OMW in a mineralization experiment and ascribed the effect to the acidic nature of OMW. However, none of the OMW treatments were acidic and neither was the soil.

3.2. Crop Response to Fertilizers

3.2.1. Crop Yield

In the first growing season, all of the organic treatments, except T1-C, performed significantly better than control in both fresh and dry weight. The best yield result corresponded with inorganic treatment IN200 followed by IN100. This suggests that the availability of nutrients in OMW compost treatments was similar to other organic treatments (vermicompost and manure), but was surpassed by inorganic treatments, as the available nutrients are related to yield [42]. After the second growing season, the dry weight of organic treatments (T1-C, T1-B, T2-C, T2-B, was slightly higher, but still statistically similar to the control. Even as the dry weight of all organic treatments was similar to the control, some treatments underperformed, such as T3-B or manure which were the lowest; and some others overperformed, such as T4-B and T4-C, which caused the highest dry weight of the overall organic treatments (Figure 1). Hernández et al. [43] reported a significantly higher yield with manure compost application in two consecutive crops of lettuce when compared with inorganic fertilization, but in this study the N rate applications of compost were 1.8 g N/pot versus 0.5 g N/pot in inorganic fertilization. On the other hand, in a long-term study under field conditions, de Sosa et al. [38] reported no detected effect in yield of olive production and olive tree growth when comparing the application of urban compost with the application of inorganic fertilization.

3.2.2. Crop Development

After 10 days of the beginning of the first growing season, there were no differences found in CCC between the treatments and the control without fertilization, only IN100 presented a significantly higher content of chlorophyl, while vermicompost and T1-C showed a negative influence during the initial growth of seedling. After 25 days, IN100, IN200, vermicompost, T3-C and T4-B showed statistically higher CCC than the control, while all other treatments presented higher values than control but without a statistically significant difference. At harvest (45 days) no difference was found in CCC between any treatment and the control except for IN200, IN100 and T2-C. All treatments showed similar evolution for chlorophyll content, although with differences between them. All fertilization treatments, specially IN100 and IN200, induced a rapid increase (0–25 days) in the chlorophyl content of leaves, and then a marked decline was observed until the harvest. The decrease observed from day 25 until the harvest was as follows: IN100 −34.1%, IN200 −33.7%, vermicompost −16.4%, and manure −9.2%, and the average value of the overall compost was −9.0%. This parameter provides information about the nutritional status of the plant and is correlated with its leaf nitrogen content [44]. The observed behavior could give an idea of how nutrients are available in the different treatments tested in this work. Inorganic fertilizers present a nutrient content readily available to plants from the first stages of growth, while compost presents a behavior similar to vermicompost or manure, stimulating the growth of plants in intermediate or senescence stage. El Hayany et al. [45] have reported that the application of compost tea and humic substances has a lower stimulatory effect on plant growth during the initial and intermediate stages, whereas in the final stage it shows significant positive effects on biometric and physiological properties, such as biomass and chlorophyll production.

In the second growing season for the first 10 and 25 days, only IN200 showed higher CCC than the control. After 45 days, inorganic treatments (IN100 and IN 200), manure, T1-B, T3-C and T4-B surpassed the control. All other treatments were higher to the control. However, due to the wide dispersion of data during this stage of growth, significant statistical differences were not obtained. The observed dynamics suggest that, except for IN200, the residual nitrogen of the other treatments was not sufficient to cover the crop demand until harvest. These results correlate with the yield results.

Regarding the dynamics of fCover, the first growing season showed no statistical difference in the first 10 days. After 25 days, every compost treatment, except T2-B, displayed a similar increment on the fCover than manure and a slightly lower one than vermicompost, while inorganic treatments caused the highest stimulatory effect on plant growth. 45 days after the beginning of the growing season, all treatments obtained significant enhancement in the green cover with respect to control treatment. The statistical study established three different groups; control treatment, all Organic treatment and IN100 and finally IN200 presented the highest green cover development.

After 10 days in the second growing season, every treatment except IN200 had no effect on the fCover compared to Control. After 25 days, T2-B showed statistically significant higher fCover than the control. All other composts were statistically similar to vermicompost, manure and the control. By the end of the second growing season, the previous behavior persists (

Table 4. Chlorophyl content (CCC) in leaves and the green cover factor (fCover) of lettuces at each moment during the growing season depending on the treatment applied to soil.

Treatment	CCC (SPAD)			fCover (%)
	10 Days	25 Days	45 Days	10 Days	25 Days	45 Days
First Growing Season
Control	37.5 ab	35.1 a	39.3 a	3.18	5.43 a	5.94 a
IN100	49.6 b	70.1 cd	46.2 ab	4.29	11.7 c	7.85 ab
IN200	42.8 ab	80.1 d	53.1 b	4.52	15.0 d	12.7 b
Vermicompost	29.9 a	54.3 bc	45.4 a	3.11	8.03 c	8.27 ab
Manure	36.7 ab	50.2 ab	45.6 a	9.32	7.75 bc	6.60 ab
T1-C	32.9 a	45.1 ab	41.1 a	3.2	6.74 abc	7.63 ab
T1-B	38.5 ab	48.3 ab	42.0 a	3.06	6.75 abc	7.16 ab
T2-C	42.8 ab	41.5 ab	46.2 ab	3.84	6.11 ab	8.47 ab
T2-B	36.7 ab	46.4 ab	42.8 a	2.73	5.79 a	8.53 ab
T3-C	41.2 ab	55.8 bc	41.8 a	3.06	7.91 bc	8.25 ab
T3-B	39.4 ab	46.8 ab	40.3 a	2.78	6.60 abc	7.19 ab
T4-C	41.9 ab	45.4 ab	44.8 a	3.06	6.66 abc	7.10 ab
T4-B	38.9 ab	52.5 bc	45.4 a	3.37	7.14 abc	7.36 ab
F-Anova	1.98 *	6.09 ***	3.48 ***	1.74 ns	36.1 ***	8.47 ***
Second Growing Season
Control	26.4 a	32.2 bcde	22.1 a	2.03 a	3.10 a	3.18 a
IN100	25.9 a	27.0 a	28.4 b	1.82 a	3.00 a	3.03 a
IN200	42.1 b	42.8 f	28.2 b	4.19 b	6.57 c	5.50 c
Vermicompost	29.4 a	30.4 abc	25.6 ab	2.57 a	3.45 ab	3.59 ab
Manure	26.6 a	33.8 bcde	29.0 b	2.21 a	3.17 a	3.67 ab
T1-C	29.0 a	31.1 bcd	26.6 ab	2.46 a	3.62 ab	3.88 ab
T1-B	26.6 a	34.3 cde	27.5 b	1.77 a	2.84 a	3.89 ab
T2-C	28.6 a	34.5 de	26.7 ab	2.42 a	3.72 ab	3.44 ab
T2-B	28.4 a	35.2 e	25.9 ab	2.58 a	4.31 b	4.16 b
T3-C	24.3 a	31.9 bcde	28.9 b	2.18 a	3.06 a	3.12 a
T3-B	28.5 a	33.2 bcde	26.1 ab	2.21 a	3.58 ab	3.54 ab
T4-C	24.9 a	30.2 ab	26.4 ab	2.39 a	3.50 ab	3.72 ab
T4-B	26.8 a	31.9 bcde	27.7 b	2.26 a	3.26 ab	3.47 ab
F-Anova	10.5 ***	14.7 ***	2.30 *	8.18 ***	14.0 ***	7.68 ***

*, ***: significant difference between treatments at p < 0.05, and p < 0.001, respectively; ns: not significant. Different letters within a column indicate significant differences between treatments (p < 0.05).

). The development of plant cover was scarce compared with the first growing season, which corroborates the idea of nutrient depletion during this second growing season. There was no evidence to suggest any negative effect on development caused by compost, as all OMW treatments showed the same or higher CCC and fCover than the control in each sampling and growing season, similar to other organic treatments and IN100.

3.2.3. Nutrient Use Efficiency

In the first growing season, crops with OMW compost application displayed similar N uptake than crops with vermicompost and manure application and all of them were above that of the control, except T1-C (Figure 2). This difference might be caused by the availability of the N in each treatment, as the N dosage was equal for all treatments (200 kg/ha) and inorganic fertilizers have higher availability and use efficiency of N [46]. The P uptake of the crops was higher in inorganic treatments (IN 100, IN200) followed by vermicompost and manure. If we compare the compost treatments with vermicompost and manure, only T1-B showed similar values of P uptake (Figure 3). Despite that, vermicompost and manure treatment reached highest P uptake values, and it is interesting to highlight that all composts had a higher phosphorus content than manure and that T1-B, T3-C T3-B, T4-C and T4-B had a higher phosphorus content than vermicompost. However, during the second growing season, a different pattern was observed: the P uptake from the compost was higher than that from the vermicompost, and, in most cases, the compost also outperformed the manure (T1-C, T2-B, T3-C, T4-B, and T4-C). Therefore, it appears that OMW composts represents a valuable source of N available for plant nutrition in the short term and also a valuable medium-term P source.

Crops treated with OMW composts showed similar K uptake to vermicompost, manure and the control, being surpassed by inorganic treatments (Figure 4). Paradoxically, Kava was higher in soils with OMW treatments than inorganic treatments. As Yin et al. have reported, higher concentrations of K in the fertilizer can decrease the use efficiency of the nutrient [47]. Additionally, K fertilization imbalances can lead to leaching of the soluble K, preventing the absorption by the plant [47,48].

After the second growing season, N uptake of all crops was very similar, with IN200 and T4-B soils increasing the uptake significantly above the control values (Figure 2). The depletion of N stock in soil from the first growing season could have led to smaller differences between treatments, as the N concentration in soils are closer to that of the control. Lastly, only IN200 led to significant changes to the K uptake of the plants at the end of the second growing season, despite the high Kava present in OMW treatment soils by the end of the second growing season (Figure 4).

The NUE of all OMW composts and organic treatments was similar. Only inorganic treatments showed statistically higher NUE, with both treatments around 60%. The average NUE of OMW treatments ranged between 9.49% and 15.0%, and was statistically similar to vermicompost and manure, suggesting equal performance than conventional organic treatments. Salim et al. [49] mentions that fertilizers with lower N content tend to have higher nutrient use efficiency. Additionally, Agegnehu et al. [50] have reported that organic treatments displayed similar NUE on the growth of barley straw between organic treatments, with better performance when paired with inorganic materials to act as organo-mineral fertilizers.

The composts showed no different NUE depending on their ingredients. However, the bulking agent used does apparently show some differences; OMW composts prepared with Alm reached lower NUE, while composts prepared with Vn reached a higher NUE. Biochar caused no statistical difference between the NUE of the OMW composts (

Table 5. Differences in NUE of the first growing season between treatments.

Treatment	NUE (%)
Treatments
IN100	60.0 b
IN200	62.7 b
Vermicompost	15.9 a
Manure	19.6 a
T1-C	13.7 a
T1-B	11.6 a
T2-C	12.6 a
T2-B	14.0 a
T3-C	9.49 a
T3-B	9.79 a
T4-C	13.4 a
T4-B	15.0 a
F-Anova	24.4 ***

CM: cattle manure; GM: goat manure; Alm: almond tree pruning; Vn: vineyard pruning. **, ***: significant difference between treatments at p < 0.01, and p < 0.001, respectively; ns: not significant. Different letters within a column indicate significant differences between treatments (p < 0.05).

). This contrasts with the reports of Agegnehu et al. [50], as they found biochar and co-composted biochar performed better than just compost. This might be due to the lower biochar concentration in the composts tested in this study.

3.3. Phytotoxicity Test of Compost

The calculated values of LC50 (related with seedling emergence) were 65.5% for compost T1, which indicates that this compost applied at this concentration can provoke a lethal effect on 50% of seeds (Figure 5), while for compost T4 the LC50 was found at 75.7% of compost concentration (Figure 6). In addition, T1 compost showed increments in the germination until a concentration of 29.2% was reached, while T4 showed a phytostimulant effect on seed germination until a concentration of 42.6% in the substrate was reached, which indicates the greater toxicity of compost T1. The EC50 values were not found in either compost T1 or T4, which indicates that the use of these composts does not cause a harmful effect on seedling growth, even at 100% compost concentration. Total dry weight increased after 12.5% and did not decrease until after 25%. Furthermore, root dry weight was always higher than aerial, suggesting the absence of inhibitory effects and potential phytostimulant for root growth. As Muscolo et al. [51] have discussed, dilution is one of the main strategies by which to decrease OMW phytotoxicity and preliminary treatments can cause significant differences in yield, supporting the idea that compost could have improved the OMW characteristics as a pretreatment, as well as diluting the OMW. Alvarenga et al. [52] have reported EC50 values, for a compost from the organic fraction of municipal solid waste, of 70.0% for barley (Hordeum vulgare L.) and 39.9% for cress (Lepidium sativum L.), justifying the idea that high compost salinity is the main limiting factor because it generates phytotoxicity in some plants [53].

4. Conclusions

Composting has been shown to be an effective treatment by which to solve the environmental issues associated with OMW toxicity. The results of this study demonstrate that composts derived from olive mill wastewater (OMW) can be safely used as organic amendments. Their application improved soil fertility by increasing oxidizable carbon, total Kjeldahl nitrogen, available phosphorus, and potassium. OMW composts achieved crop yield and plant development comparable to common organic treatments such as vermicompost or fresh manure and provided a valuable source of nitrogen for short-term plant nutrition and a medium-term source of phosphorus. Phytotoxic effects were not observed, as germination and growth tests confirmed the absence of inhibitory impacts. Even proportions <30% showed phytostimulant effects, especially on germination and root growth. Field studies, as well as in situ composting treatment, should be further evaluated; however, the overall results obtained in this work demonstrate that OMW composting represents a sustainable strategy by which to valorize this residue and to contribute to both soil health and the circular economy.