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253
received: 2019-05-23                  DOI 10.19233/ASHN.2019.27
DETERMINATION OF HEAVY METAL CONTENT IN A DAPHNE GNIDIUM L. 
PLANT USING ATOMIC ABSORPTION SPECTROSCOPY
Jamila FLIOU, Ali AMECHROUQ, Mohammed ELHOURRI, Ouassima RIFFI & Mostafa EL IDRISSI
Laboratory of Molecular Chemistry and Natural Substance, Moulay Ismail University, Faculty of Science, 
B.P. 11201 Zitoune, Meknes, Morocco
e-mail: flioujamila@gmail.com 
ABSTRACT
The objective of this study was to determine the content of heavy metals in a Daphne gnidium L. plant col-
lected in the Middle Atlas (Ribat El Kheir). This plant is used in traditional medicine for its antioxidant effects and 
antigenotoxic, antiseptic and healing properties. This study is partly based on a differential thermal analysis (DTA), 
which indicated mass losses in three temperature regions. To explain this phenomenon, a sample of D. gnidium 
was heated in an oven at different temperatures: 100 °C, 300 °C and 600 °C for 6 hours, and after the calcination 
the sample was analysed by Fourier-transform infrared spectroscopy. The results of the study show that the plant D. 
gnidium contains several metals, with increased levels of Fe and Mg. These values are alarming because they are a 
major danger to the users of this plant.
Key words: Daphne gnidium, heavy metals, AAS, DTA, GTA 
DETERMINAZIONE DEL CONTENUTO DI METALLI PESANTI IN PIANTE DI DAPHNE 
GNIDIUM L. CON L’USO DI SPETTROSCOPIA DI ASSORBIMENTO ATOMICO
SINTESI
Lo studio si prefiggeva di determinare il contenuto di metalli pesanti in piante di Daphne gnidium L. raccolte nel 
Medio Atlante (Ribat El Kheir). Questa specie è utilizzata nella medicina tradizionale per i suoi effetti antiossidanti 
e le proprietà antigenotossiche, antisettiche e curative. La ricerca in parte si basa su un’analisi termica differenziale 
(DTA), che ha indicato perdite di massa in tre regioni di temperatura. Per spiegare questo fenomeno, un campione 
di D. gnidium è stato riscaldato in un forno a diverse temperature: 100 ° C, 300 ° C e 600 ° C per 6 ore, e a seguito 
della calcinazione il campione è stato analizzato mediante spettroscopia in trasformata di Fourier. I risultati dello 
studio mostrano che D. gnidium contiene diversi metalli, con alti livelli di Fe e Mg. Questi valori sono allarmanti 
perché rappresentano un grave pericolo per l’utilizzo di questa specie a scopo curativo.
Parole chiave: Daphne gnidium, metalli pesanti, AAS, DTA, GTA
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INTRODUCTION
For a long time, people have used plants for their 
medicinal properties (Ivanova et al., 2005), and in recent 
years the search for new substances based on natural 
products has increased. These can be beneficial to 
humans as natural cures for certain diseases (Roh et al., 
2004). The subject of this study is Daphne gnidium L., 
a spontaneous plant species common in semi-arid and 
subhumid areas of the Mediterranean region. The flow-
ers appear during March–October. It is mainly used in 
traditional medicine as a remedy for hepatitis, toothache 
and skin diseases; the methanol extract of D. gnidium 
is known for its antibacterial and antifungal properties 
and good skin tolerance (Iauk et al., 1996; Iauk et al., 
1997). Unfortunately, the industrial development and the 
introduction of new inputs into agriculture, such as ferti-
lizers and pesticides, which can be transported by wind 
or water, have compromised the quality of these plants, 
some of which are now becoming unfit for human use 
due to the concentrations of heavy metals they contain. 
Today, much research is directed towards the study of the 
impact of heavy metals (Cd, Cu, Pb, Zn) on the germina-
tion and growth of plants. Mihoub et al. (2005) showed, 
during the germination of pea beans (Pisum sativum L.), 
that the cotyledons in stressed grains gradually accumu-
late Cd and Cu and retain high levels of Fe, Mg, and 
Zn. Remarkably, under conditions of heavy metal stress, 
some higher plants are able to focus the excess metal in 
certain organs (Sbartai et al., 2012). Several factors may 
be responsible for the content of heavy metals in plants: 
the solubility of metals in the soil, soil properties (pH, 
redox potential), climate change, fertilizers, pesticides, 
and root systems (Kabelitz & Barbin, 1999). Khan et al. 
(2008) have shown that large amounts of heavy metals, 
such as Ni, Cd, Cr and Pb, are introduced into the soil 
through agricultural and industrial activities.
Other studies have shown the influence of tem-
perature on the release of Cd, Pb, Cu, and Zn from the 
branches and leaves of Populus nigra L. at temperatures 
ranging between 653 and 873 °K, in conditions of slow 
steam-assisted pyrolysis (Grottolaa et al., 2019). 
While some metals may exist naturally in plants 
(Ca, Fe, Na, etc.), others are present in the plant her-
itage because of air pollution, agricultural activities 
and industrial discharges (Al, Cd, Pb). Several studies 
have been conducted with the prospect of their results 
proving useful to the development of phytoremediation 
techniques to decontaminate the soils of abandoned 
industrial sites (Garcia et al., 2003). Although many 
metals, such as copper (Cu), iron (Fe), manganese (Mn), 
nickel (Ni), and zinc (Zn), are essential for cells in low 
concentrations, but toxic in high amounts (Millaleo et al. 
2010), there are also metals that have not been proven 
as necessary for the development of living organisms 
and are toxic even in very low concentrations (Hg, Pb, 
Cd) (Todeschini et al., 2011).
MATERIAL AND METHODS
Plant material
The sample of D. gnidium was harvested from Ribat 
El Kheir (33° 49’ N, 4° 25’ W) located in the Middle Atlas 
of Morocco (Fig. 1). The climate in this region is varied, 
but experts consider it Mediterranean: average annual 
temperature 28 °C, maximum temperature close to 43 
°C, minimum temperature between 4 and 6 °C, frosts 
observed in January and February. The annual rainfall av-
erage is 500 mm. Fog is infrequent. The prevailing winds 
are from east to southwest or from southwest to northeast.
DTA/TGA
Five milligrams of D. gnidium powder were used to 
conduct a differential thermal analysis (DTA) with a 60 
Shimadzu workstation, performing simultaneously DTA/
TGA at a heating speed of 20 °C/min for 29 min15s; note 
that this process is linear. DTA is generally associated 
with thermal gravimetric analysis (TGA), which meas-
ures mass loss versus temperature. This analysis deter-
mines the temperature intervals in which the sample 
undergoes changes due to the heat treatment applied. 
The results of DTA are presented in the form of a curve: 
DT (in microvolts) = f (T) (in °C). 
Infrared
The same sample was analysed, in the presence of 
ground potassium bromide, with the Fourier-transform 
infrared spectroscopy (FTIR) technique, using a JASCO 
4100 device and recording spectra over the range of 
4000 cm-1 to 500 cm-1.
Atomic absorption spectroscopy (AAS)  
The ash recovered after the calcination of the plant 
at 600 °C in a muffle furnace, was mineralized with 
Fig. 1: Geographical map of the region of Ribat El Kheir.
Sl. 1: Zemljevid območja Ribat El Kheir.
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concentrated nitric acid and filtered with Whatman 0.45 
mm filter paper to obtain a solution ready for the deter-
mination of metals by atomic absorption spectroscopy.
The AA-7000 series of atomic absorption spectro-
photometers produced by the Japanese group Shimadzu 
is able to measure many different metals. It is composed 
of an atomizer, which prepares the sample for analysis, 
a radiant lamp, and a detector. The detection limit in this 
case was 0.03 ppb. The result of the AAS is presented 
in the form of a curve: A=f(C), where A = absorption of 
metal, and C = concentration of metal in ppm
RESULTS AND DISCUSSION
Before performing DTA, we analysed the raw plant 
by infrared spectroscopy (Fig. 2). The infrared spectrum 
showed a frequency around 3437 cm-1 dominated by 
the vibration n(OH) of aromatic and aliphatic structures, 
and a band around 2925 cm-1 corresponding to the va-
lence vibration strip n(C-H). The spectrum also showed 
a defined band at 1630 cm-1 that could be assigned to 
the valence vibration n(C=C) of the aromatic structure. A 
remarkable band at a frequency of 1382 cm-1 was attrib-
uted to the n(C-O-C) vibration of aryl-alkyl ether. The 
band that appeared around 1038 cm-1 indicated the 
presence of a (Si-O) group (Fig. 2). 
In order to understand the molecular composition of 
the plant, 5 mg of D. gnidium were used for a thermal 
analysis (DTA). The results of this analysis are shown in 
Figure 3. The DTA allowed us to identify three tempera-
ture intervals at which we observed significant mass loss. 
As shown in Table 1, significant losses of mass were 
observed from 160 °C onwards, and all of the organic 
material was lost at 600 °C.
To explain this weight loss at different temperature 
ranges, the sample was heated in a muffle furnace at 
different temperatures: 110 °C, 300 °C and 600 °C for 
calcination. After cooling in a desiccator, the samples 
were analysed by IR spectroscopy (KBr). Table 2 shows 
mass loss at each temperature range.
The results of the calcination confirmed those of 
the DTA. We noted that more than half of the organic 
material was lost at 300 °C, and another important loss 
occurred at 600 °C.
Figure 4 shows the superposed IR spectra of the 
calcined samples. At 110°C, we noted the persistence 
of vibration valence bands of the n(OH), n(C-H), and 
n(C=C) bonds. At 300°C, we noted the disappearance 
of (OH), the appearance of a vibration band of the 
 
 
Fig. 2:  
 
Fig. 2: IR spectrum of D. gnidium.
Sl. 2: IR spekter vrste D. gnidium. 
 
Fig. 3:  
 
Fig. 3: DTA/TGA spectrum of D. gnidium.
Sl. 3: DTA/TGA spekter vrste D. gnidium. 
Tab. 1: Mass losses of D. gnidium in DTA.
Tab. 1: Masne izgube vrste D. gnidium na diferencialni 
termalni analizi (DTA).
Temperature (°C)
Mass losses 
in %
Total losses (%)
0-160 °C 11.24% 11.24
160-365 °C 46.19% 57.43
365-600 °C 18.14% 94.68
Tab. 2: Mass losses in the calcining D. gnidium (mi: the 
initial mass, mf: the final mass).
Tab. 2: Masne izgube pri žganju vrste D. gnidium (mi: 
začetna masa, mf: končna masa).
 
Temperature (°C) mi (g) mf (g) Loss (%)
110 5.44 4.66 14.34
300 5.32 2.20 58.64
600 10.73 1.11 89.65
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n(N-H) bond at 3436 cm-1 corresponding to secondary 
amines, this latter was accompanied by the appearance 
of a deformation valence band of the d(N-H) bond. The 
spectrum also recorded decreases in band intensities of 
n(C-H) and n(C=C) bonds, which indicated the begin-
ning of disappearance of the organic matter.
At 600 °C, we noticed the disappearance of the 
vibration band of the n(N-H) bond and the band related 
to organic matter, and bands related to other mineral 
elements, such as kaolinite, smectite, quartz, and calcite 
(Fig. 5) appearing instead. These results were in good 
agreement with the literature (Hachi et al., 2002).
The sample that was calcined at 600 °C and treated 
with concentrated nitric acid was subsequently analysed 
by AAS. The results showed a very high Fe and Mg 
content (1207.7906 mg/kg). These values are alarming, 
indicating a serious health threat to potential users of this 
plant (Koumolou et al., 2012). Sathiamoorthy et al. (2003) 
conducted comparative studies on 42 medicinal plants 
from the Negev desert. The highest iron concentrations 
were recorded in Gundelia tournefortii (3020 ppm) and 
Anchusa strigosa (2485 mg/kg). The concentration of Cu 
in D. gnidium was 0.2738 mg/kg. This value is below the 
limit concentration in raw plant material provided by the 
World Health Organization (WHO), which is 150 mg/
kg (World Health Organization, 2007). The value of Li 
was 0.0083 mg/kg. These values show that the copper 
and lithium levels in the plant posed no risk for its users.
In relation to Zn, the limit value in D. gnidium was 
1.7683 mg/kg), which is within the standards (1 to mg/
kg). This means that in terms of Zn, the plant was not 
a significant threat to its consumers, who use it as an 
antiseptic or healing agent (Koumolou et al., 2012). Not 
so the leaves of Populus euphratica and Z. geslini from 
the mentioned comparative study, which registered Zn 
concentrations well above the norm: 113 mg/kg and 
119.10 mg/kg respectively (Sathiamoorthy et al., 2003).
While the concentration of Pb in D. gnidium was 
0.5148 mg/kg – below the WHO limit (10 mg/kg) (Boulila 
Zoghlami et al., 2006), Cd concentration was 0.3787 mg/
kg – exceeding the WHO tolerance limit (0.3 mg/kg) by 
26.23% (World Health Organization, 2007). Regarding 
Al, atomic absorption spectroscopy showed that it was 
not present in D. gnidium, while the value for Ca was 
32.5020 mg/kg. Studies conducted by Boulila Zoghlima 
et al. (2006) on tomato (Lycopersicon esculentum) showed 
that the negative impact of cadmium on certain growth 
and development processes can be mitigated by an ad-
equate calcium intake in the culture medium, and that the 
addition of Ca to a culture medium containing Cd (growth 
inhibitor) improves the production of plant biomass.
CONCLUSIONS
In the present study, we have tried to assess the toxic-
ity of the plant D. gnidium through the determination of 
the heavy metals in it that could harm the health of the 
plant’s consumers. The results of the analyses conducted 
revealed high concentrations of Fe, Mg, and Cd, thus 
confirming the concerns which had prompted this study. 
This contamination by heavy metals could be the cause 
of many diseases among the population consuming this 
plant, hence the need to seek the origin of these metals 
(soil, water, agricultural activities) and of the process of 
contamination, especially since D. gnidium bark pow-
der is also used orally in the treatment of syphilis and 
venereal diseases.
Fig. 4: Superposed IR spectra of the samples carried on 
ignition.
Sl. 4: Krovni IR spekter žganih vzorcev. 
 
 
 
 
Fig. 4:  
 
Fig. 5: IR spectrum of the sample calcined at 600 °C 
(K: kaolinite, S: smectite, CA: calcinite, Q: quartz, MO: 
organic material).
Sl. 5: IR spekter vzorca segretega na 600 °C (J: kaolinit, 
S: smektit, CA: kalcinit, Q: kremen, MO: organska snov).
 
 
 
Fig. 5:  
 
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DOLOČEVANJE VSEBNOSTI TEŽKIH KOVIN V VOLČINU VRSTE DAPHNE GNIDIUM L. 
Z UPORABO ATOMSKE ABSORBCIJSKE SPEKTROSKOPIJE
Jamila FLIOU, Ali AMECHROUQ, Mohammed ELHOURRI, Ouassima RIFFI & Mostafa EL IDRISSI
Laboratory of Molecular Chemistry and Natural Substance, Moulay Ismail University, 
Faculty of Science, B.P. 11201 Zitoune, Meknes, Morocco
e-mail: flioujamila@gmail.com 
POVZETEK
Cilj raziskave je bil določiti vsebnost težkih kovin v volčinu vrste Daphne gnidium L., nabranem v osrednjem 
Atlasu (Ribat El Kheir). To rastlino uporabljajo v medicini kot antioksidant in zaradi antigenotoksičnih, antiseptičnih 
in zdravilnih učinkovin. Raziskava  delno temelji na diferencialni termalni analizi (DTA), ki pokaže izgubo mase v 
treh temperaturnih regijah. Da bi razložili ta pojav so vzorec vrste D. gnidium segrevali v pečici na različnih tempe-
raturah: 100 °C, 300 °C in 600 °C za 6 ur, in ga po segrevanju analizirali s Fourierjejevo infrardečo spektroskopijo. 
Rezultati so pokazali, da vsebuje volčin številne kovine, še posebej visok nivo Fe in Mg. Te vrednosti so alarmantne, 
saj lahko povzročijo nevarnost pri uporabnikih. 
Ključne besede: Daphne gnidium, težke kovine, AAS, DTA, GTA 
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