*3.2. Rainfall Runoff Trial*

The plant dry matter displayed a strong positive, linear relationship with the plant cover (Figure 3; *plant cover* = 0.626*DM* + 1.95; *r*<sup>2</sup> = 0.94). The treatments receiving inorganic N fertiliser in the form of (NH4)2SO4 (ASLow, ASMedium and ASHigh) contained significantly lower plant dry matter and percentage cover than the treatments receiving the combined ((NH4)2SO4 + CropUpTM) fertiliser (CULow, CUMedium and CUHigh). The ammonium sulphate treatments produced a plant cover of approximately 40–45%, while the combined (NH4)2SO4 + CropUpTM produced plant covers ranging from 70 to 90% (Figure 3). The lower plant cover was consistent with the relatively poor ryegrass (*Lolium multiflorum*) seed germination observed during the 42-day growth stage.

**Figure 3.** Effect of treatment on: (**a**) percentage plant cover; (**b**) plant biomass; and (**c**) plant total N. Bars represent the mean of three replicates (*n* = 3) and bars designated by the same letter are not significantly different at (*p* < 0.05).

The ryegrass treatments (CULow, CUMedium and CUHigh) contained significantly (*p* < 0.05) more plant C and plant N than ASLow, ASMedium and ASHigh (Table 4 and Figure 3). The plant macronutrient (P, K, Ca, Mg and S) and micronutrient (Al, B, Fe, Mn, Na and Zn) content was significantly higher for the ryegrass grown in the presence of CropUpTM compared with the control and (NH4)2SO4 treatments (Table 4).


*Nitrogen* **2022** , *3*

The nitrogen use efficiency (NUE) was estimated as the proportion of applied N taken up by the ryegrass (Table 4). The NUE for the (NH4)2SO4-only treatments ranged from 58% to 75% in the (NH4)2SO4 + CropUpTM treatments, and was two-fold greater than for the (NH4)2SO4) treatment (24–42%).

The masses of total N, NH4, NOx and mineral N (NH4 + NOx) in the runoff from the Control, ASLow, ASMedium, ASHigh, CULow, CUMedium and CUHigh treatments are presented in Figure 4. The masses of runoff total N, NH4 and mineral N for the inorganic N fertiliser ((NH4)2SO4) were higher (*p* < 0.05) than for the (NH4)2SO4 + CropUpTM treatments. For example, ≥200 mg of total N was collected for the inorganic N treatments compared with ≈100 mg for the (NH4)2SO4 + CropUpTM treatments, which represented a two-fold reduction in N runoff. The runoff NO3 content was very low for all treatments.

**Figure 4.** Masses of (**a**) total N, (**b**) NOx, (**c**) NH4 and (**d**) mineral N (NH4 + NOx) in runoff from the Control, ASLow, ASMed, ASHigh, CULow, CUMed and CUHigh treatments. Bars represent the mean of three replicates (n = 3). Same lowercase alphabets indicate statistically non-significant differences between treatments at *p* < 0.05.

## **4. Discussion**

#### *4.1. Nitrogen Release from Organic and Inorganic Sources*

Leaching with dilute 0.005 M CaCl2 displaced >99% of the N added as NH4Cl but only 15% of that added as CropUpTM, with NH4 being the dominant form of mineral N in the leachate. This low recovery of mineral N from CropUpTM results from the slow rate of nutrient release from this organic material (i.e., 0.3 d−1) relative to readily available inorganic substrates (i.e., 0.9 d<sup>−</sup>1). Importantly, this slow rate of release of N from CropUpTM would be highly beneficial for the nutrient dynamics in terms of optimising N availability relative to plant uptake requirements and reducing the N loading in the soil solution where it is susceptible to leaching and runoff loss, such as in sandy soils. Furthermore, although the leachate NO3 concentrations were very low for all N substrates, CropUpTM tended to encourage nitrification relative to the control and NH4Cl treatments, possibly through the introduction of nitrifying bacteria within its organic substrate, or by stimulating the resident, albeit limited, nitrifying bacteria population within the sand. Stimulating the microbial community in terms of functionality and diversity is highly beneficial in sandy soil, particularly in terms of nutrient cycling and immobilisation, which can act as a mechanism for nutrient storage and subsequent release, and for reducing leaching losses in low-cation exchange capacity (CEC) materials [33–35].

The loss of mineral N primarily as NH4 demonstrates the sand's poor ability to retain this cation, thereby encouraging potential losses through leaching and runoff. The cation exchange capacity (CEC) of the sand is 1.1 cmol+/kg (equivalent to 6.16 mg of negative charge per 40 g of sand expressed as N (Table 1)). The 0.005M CaCl2 leaching solution provided a ready supply of Ca capable of competing with NH4 for the limited cation exchange sites. Over the leaching study, approximately 0.35 mmoles of Ca was added to the sand, being equivalent to ≈10 mg of N. This mass of Ca exceeded the amount of negative charge (6.16 mg) available to retain the 9 mg of N added in the NH4Cl and CropUpTM treatments. Furthermore, the rapid loss of NH4 from the sand during the early leaching events suggests that the sand's cation exchange sites may have a stronger preference for Ca relative to NH4, thereby favouring NH4 displacement and leaching with the 0.005 M CaCl2 solution. Although this effect was more pronounced for the NH4Cl treatment, similar behaviour may be expected for the CropUpTM treatment. Therefore, the extremely low CEC of the sand coupled with a higher preference for Ca may explain the similar 2M KCl-NH4 concentrations measured between treatments (i.e., 0.5 cmol+/kg). The poor NH4 retention by sandy soils would not only encourage N leaching, but would also result in a loss of this nutrient in runoff and lateral soil water flow (see below Rainfall runoff trial).

The mass of mineral N recovered from the CropUpTM and NH4Cl treatments in excess of the control was 1.04 and 9.45 mg, respectively (Table 3). Given each treatment received 9.72 mg of N, the percentage recovery of N added to the CropUpTM and NH4Cl treatments was approximately 15% and 100%, respectively. This suggests that nearly all of the N added as NH4Cl but only 15% of that added in CropUpTM was readily available over the 42-day study (Table 3). The mineralisation rates for pelleted poultry manure have been reported to be approximately 10%, 23% and 36% of initial N (total N = 2–4%) after 1, 4 and 8 weeks' incubation (25 ◦C), respectively [36]. These mineralisation (or N release) rates are not dissimilar to those reported in this study and show CropUpTM as a supplementary N source to inorganic fertiliser, which would have been most beneficial for ryegrass establishment and sustainability during the latter stage of the 42-day growth period when inorganic N sources had been depleted and the grass growth rates were high.

CropUpTM contains approximately 4500 and 40 mg/kg of 2M KCl extractable NH4 and NOx, respectively. Of the 9.72 mg total N added in 0.347 g of CropUpTM, 1.56 mg was as NH4, and 0.01 mg as NOx. Therefore, approximately 8.1 mg of N was present in non-mineral forms, probably as organic N. Over the 42-day leaching study, approximately 1.44 mg of mineral N (NH4 + NOx) was recovered (>92%; Table 3), which was very similar to that added in CropUpTM. This shows that leaching with 0.005 M CaCl2 effectively removed mineral N from CropUpTM with negligible contributions from other fractions, such as organic matter. CropUpTM has a C:N ratio of 7.6 and a total N content of 3%, which may be expected to encourage the mineralisation of organic N to NH4 [37]. The sand may therefore lack a significantly large functional microbial community capable of organic matter mineralisation and subsequent nitrification. Limited nitrification in the sand is supported by the absence of NO3 in the NH4Cl treatment (Figure 1). However, organic inputs to sands have been shown to steadily increase organic matter and to alleviate poor microbial activity [12,19,33].
