1.1 Composition of livestock wastewater
Table 2
The total HMs concentrations in livestock farm effluent and their impact to the human health, and the maximum concentration levels of those HMs in quality standards B.
Heavy metals (HMs) | Concentrations (mg/L) | Maximum concentration levels (MCL) of heavy metals Standard B (mg/L) |
Cr | ND | 0.05 |
Pb | ND | 0.5 |
Mn | 2.2 | 1.0 |
Ni | ND | 1.0 |
Cu | 0.121 | 1.0 |
Zn | 0.677 | 2.0 |
As | ND | 0.1 |
Al | 0.699 | 15.0 |
TP | 97.7 | ND |
TN | 164.6 | ND |
1.2 Atomic Absorption of spectrophotometer (AAS) for manganese concentration in L. minor and A. pinnata
1.2.1 Removal efficiency of aquatic plants in livestock wastewater (%)
Removal of efficiency (%) =\(\frac{Ci-Ce}{Ci} x 100\)
where,
Ci = mean concentration amount of heavy metal initial
Ce = mean concentration amount of heavy metal at specific time
Table 3
The removal efficiency (RE) of Mn in aquatic plant species, including L. minor and A. pinnata, at various time exposure
Treatments | Types of aquatic plants | Exposure time |
0 hours | 24 hours | 48 hours | 72 hours |
(concentration mg/L) | (concentration mg/L) | Removal efficiency (%) | (concentration mg/L) | Removal efficiency (%) | (concentration mg/L) | Removal efficiency (%) |
Livestock Wastewater (LW) | Lemna minor | 16.6 ± 0.1 | 27.6 ± 0.1 | 66 | 53.1 ± 0.1 | 92 | 74.7 ± 0.1 | 41 |
Azolla pinnata | 21.1 ± 0.1 | 29.4 ± 0.1 | 39 | 31.4 ± 0.4 | 6.8 | 55.6 ± 0.5 | 77 |
Table 4
The correlation between the accumulation rate of L.minor and A.pinnata with exposure time
| Time | LemnaMinor | |
Time | Pearson Correlation | 1 | .989* | |
Sig. (2-tailed) | | .011 | |
N | 4 | 4 | |
LemnaMinor | Pearson Correlation | .989* | 1 | |
Sig. (2-tailed) | .011 | | |
N | 4 | 4 | |
*. Correlation is significant at the 0.05 level (2-tailed). | |
| Time | AzollaPinnata |
Time | Pearson Correlation | 1 | .918 |
Sig. (2-tailed) | | .082 |
N | 4 | 4 |
AzollaPinnata | Pearson Correlation | .918 | 1 |
Sig. (2-tailed) | .082 | |
N | 4 | 4 |
Table 5
Morphology description of L. minor and A.pinnata according to different exposure time
Types of aquatic plants | Morphological characteristics | Exposure time |
T0 | T1 | T2 | T3 |
L.minor | Leaf shape | Normal | Normal | Normal | Normal |
Root | Long | Long | Long | Long |
Color | Pale green | Light green | Light green | More light green than pale green |
A.pinnata | Leaf shape | Looks fresh, normal shape | Shrink | Shrink | Shrink |
Root | Long | Short | Short | Disappeared |
Color | Light green | Black + green | Black + green | Black + green |
1.4 Sem Observation
1.5 Genetic profile of A. pinnata and L. minor using RAPD Marker
Result And Discussion
The composition of livestock wastewater collected from sampling site showed excessive levels of the heavy metal manganese (Mn), as listed in Table 2. Manganese is one of the elements that has previously been reported to be present in cow dung (Gupta et al. 2016). In livestock production this element is an essential trace mineral known as manganese performs as both an enzyme component and an enzyme activator (Spears 2019). However, manganese toxicity can result from over consumption of this mineral which affects the central nervous system, (Sidoryk-Wegrzynowicz and Aschner 2013).
The plants were revealed to accumulate large levels of Mn in a concentration and time dependent way (Fig. 1). The elimination of the abovementioned heavy metals was equivalent to their net sorption in plants due to natural precipitation L. minor sp. showed greater sorption capability than A. pinnata. L. minor was highly efficient in absorbing heavy metals (Mn) in livestock wastewater with a RE of 92% at 48hours treatment while A pinnata shows RE of 77% at 72 hours exposure as recorded in Table 3. A significant strong positive relationship between retention time and heavy metal showed significance value p ≤ 0.05 (Table 4). According to Paolacci et al. (2021), the duckweed removal system showed larger plant densities resulted in a higher removal rate per square metre of water, increasing the number of plant’s capacity to absorb nutrients. This species has long thin root which has known to be main characteristics of greater removal hyperaccumulator in livestock wastewater. Our findings is similar to (Bokhari et al. 2016; Al-Khafaji et al. 2018), for the removal of heavy metals, with RE of > 80% which showed L. minor are an excellent candidate.
In addition to physiology capability, the morphology of the plants should be considered in order to determine their potential for RE. Visible observation of the plants indicated that healthy plants appeared to be in great health and to have a good adaptation of their surroundings. The morphology description of L.minor and A.pinnata has been recorded in Table 5. At the end of exposure period, leaves of L. minor turned pale green to yellowish color whereas those A. pinnata leaves turned into brown and black (Fig. 2). It is known that the plants leaves are more sensitive to environmental changes than its other organs (Brandão et al. 2018). The greenish colour of the plants has been turned to pale green and almost white in colour due to mechanism of chlorophyll occurred in the aquatic plants. This is similar to finding by Wang et al. (2014), the fronds of L. minor exposed to high nitrogen ammonia concentrations during the cultivation process, had a chlorotic and white colour that was gradually extending from the edge to the centre of each frond. The decrease in chlorophyll content may be caused by chloroplast membrane peroxidation or by Pb ions substituting for magnesium in chlorophyll molecules (Peng et al. 2019). Based on this study, A. pinnata could not be further applied as phytoaccumulator as this plants show sign of cell death as early as 24 hours exposure. According to Sudiarto et al. (2019), the wastewater probably possessed characteristics that prevented the plants from growing and absorbing nutrients. These results demonstrate the potential of using appropriate floating plant species in phytoremediation, as different plant species resulted in diverse removal efficiencies of Mn from livestock wastewater. A.pinnata exposed to livestock wastewater had leaf surfaces that were obviously different from the untreated control (Fig. 3). The leaf surface of A.pinnata was decreased by the livestock wastewater treatment and it may be that an excess of Manganese (Mn) resulted in shrinking. The key features of Program Cell Death (PCD) are shrinking of the protoplast and nucleus, condensation of chromatin, breaking of DNA, and vacuolization. According to Temmink et al. (2018), when maximum fixation rates by diazotrophic symbionts were reached for high P levels, the plants turned chlorotic. Evidently, the diazotrophs were unable to fix extra N that might have been used to boost biomass growth. This is in line with a study by Muradov et al. (2014), observed that Azolla plants were more sensitive to anaerobically digested swine wastewater (ADSW), becoming brown from the centre of the fronds and ultimately dying after 5 to 7 days at concentrations of 50 to 10%. Excessive nutrient in livestock wastewater lead to stressing in nutrient stoichiometry which may give death effect to the A. pinnata. According to Temmink et al. (2018), at greater P concentrations (> 50 µmol− l ) chlorosis appears to be brought on by iron (Fe-) rather than a lack of nitrogen (N-). A study by Sangwijit et al. (2021), observed that another aquatic macrophytes H. verticillata was unable to grow in university canteen wastewater (UCW) as a result of its photosynthesis being inhibited by high nutrients, turbidity, and organic substances. The morphology of the aquatic plants' roots has also been examined in this study for any alterations that might occur when exposed to livestock wastewater. Based on SEM observation root surface of the treated samples in both aquatic plants seen to shrink and shorten. Heavy metals are adsorb in cationic form with negative cell walls on the surface of the roots due to the existence of cellulose, pectins, and glycoproteins, which function as particular ion exchangers (Arif et al. 2016).
Using the RAPD technique, rearrangements, point mutations, insertions and deletions of DNA, and ploidy alterations in genomic DNA caused by genotoxic substances found in excessive levels in the environment (Erturk et al. 2013). RAPD profile in this study confirmed genotoxic effect of livestock wastewater which alter L. minor and A. pinnata genome. DNA profiles have shown increased/decreased band intensities, the emergence of new bands, and the removal of typical bands. In this study, overall seven (or 58%) of these primers produced significant band, but another 5 primers were not functional for amplification. From functional primers C17, amplified > 10000bp bands and produced four reproducible band profiles that might be used to determine treated treatments from controls (Fig. 4A). No new bands appeared or disappeared, however there was a significant increase in band intensities in A.pinnata with control and different exposure time in livestock wastewater. The band intensities was increased at T1-T3 compared to the control. The increment in band intensities has been reported to occur in DNA profiles of Urtica pilufera when exposed in different concentration of Cadmium (Cd) (Dogan et al. 2016). In another study, various band intensities was observed in DNA profile of Sphagnum palustre, suggesting similar genotoxic effect related to heavy metals (Sorrentino et al. 2017). From another functional primers, bands obtained using B07 ranged from 200 bp to 1000bp (Fig. 4B). Primer B07 produced 21 reproducible band profiles and also demonstrated that DNA fingerprints between livestock wastewater exposed and control group were clearly distinguished (Fig. 4A). The variation in band intensities were observed as decreased in 800bp T1 and T2 and increase in T3 in treated group. Moreover, new band appeared between 800bp to 1000bp in T3 was observed in L. minor. This finding is similar with Ozyigit et al. (2021), whereby a new band was found in the 200 µM Cd-Ni treatment level. A total of 19% polymorphism were detected in L.minor. The presence of polymorphism in RAPD profiles demonstrates that these plants have evolved to withstand the stress caused by the presence of heavy metals in livestock wastewater. In other study by (Swaileh et al. 2006), observed the RAPD profile of raw wastewater whereas raw wastewater is much more genotoxic than treated wastewater (TWW) and the treatment process in this treatment plant removes some, but not all, genotoxic substances from wastewater. Another study by (Yigider et al. 2016) revealed that Mn stressors caused Z. mays seeds to develop several primers known as Stowaway, Sukkula, BARE 1(0), N-57(Nikita), and Nikita-E2647 LTR retrotransposon polymorphism. In contrast to A.pinnata, although this plant did not induce any polymorphism, all treated samples at all exposure times showed increased in band intensities as compared to untreated samples. There are several studies reported that heavy metals induced plant stresses which cause retrotransposons' transcriptional levels to increase (Cong et al. 2019; Gallo-Franco et al. 2020). According to Ozyigit et al. (2019), by analyzing differences in DNA banding profiles between collected sample species using these molecular markers, it is possible to determine the damaging effects of heavy metals on the integrity of genetic material. This is due to the genetic instability of plants due to heavy metals exposure to the cells (Morales et al.2016). A few studies investigated the genotoxicity by DNA molecular markers in aquatic plants so far ( Tanee et al. 2016; Ozyigit et al. 2021), to our knowledge, this paper is the first to contribute to livestock wastewater contained excessive of Mn.