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Environmental Toxicology and Pharmacology 56 (2017) 225–232
Contents lists available at ScienceDirect
Environmental Toxicology and Pharmacology
journal homepage: www.elsevier.com/locate/etap
Research Paper
The potential effects of efavirenz on Oreochromis mossambicus after acute
exposure
L Robsona, I.E.J. Barnhoornb, G.M. Wagenaara,
a
b
MARK
⁎
Department of Zoology, University of Johannesburg, PO Box 524, Auckland Park, 2006, South Africa
Department of Zoology, University of Venda, Private Bag X5050, Thohoyandou, 0950, South Africa
A R T I C L E I N F O
A B S T R A C T
Keywords:
Pharmaceuticals in the environment
Antiretroviral drugs
HIV
Fish histology
Histopathology
Aquatic toxicology
Antiretroviral drugs (ARVs) are hazardous therapeutic pharmaceuticals present in South African surface water.
Efavirenz is an ARV commonly used in human immunodeficiency virus (HIV) treatment in South Africa.
Although little is known about the toxic effects of efavirenz on fish health, threats of toxicity to the aquatic
environment have been reported. Oreochromis mossambicus were exposed under controlled conditions to environmentally-relevant efavirenz concentrations (10.3 ng/l) as measured in rivers that flow into the Nandoni
Dam in the Vhembe District, South Africa. Acute (96 h) exposures were conducted using efavirenz concentrations of 10.3 ng/l and 20.6 ng/l. The overall health of exposed fish was determined using a histology-based fish
health assessment index. Necropsies and haematology were conducted and somatic indices calculated after
which the liver, kidney, heart, gills and gonads were microscopically quantitatively assessed. Results indicated
that fish exposed to 20.6 ng/l efavirenz had significantly (p < 0.02) higher liver indices than the control fish,
indicating increased liver damage including steatosis and frank necrosis. Fish exposed to 20.6 ng/l efavirenz
presented with significantly (p < 0.02) higher total fish indices, representative of declined overall health
compared to control fish. It was concluded that the exposure of O. mossambicus to efavirenz resulted in liver
damage and overall decline in fish health. These novel findings may indicate a health risk for O. mossambicus and
other biota exposed to efavirenz in aquatic ecosystems. Thus, ARV’s in water sources of South Africa pose a
definite threat to wildlife and ultimately human health.
1. Introduction
The human immunodeficiency virus (HIV) and acquired immune
deficiency syndrome (AIDS) are a worldwide problem, however Africa
has the highest prevalence of the disease (World Health Organisation
(WHO), 2016). South Africa has an estimated 7 million people living
with HIV, of which 3.6 are receiving antiretroviral drug (ARV) treatment thus making South Africa’s ARV treatment programme the largest
in the world (Mapumulo, 2016; UNAIDS, 2015). ARVs have recently
been recognised as emerging pollutants and have been quantified in the
aquatic environment in trace concentrations in South Africa
(Swanepoel et al., 2015; Wood et al., 2015). They are also known to be
highly toxic and are classified as an “extremely hazardous therapeutic
class” (Swanepoel et al., 2015). A report by the Water Research Commission (WRC) estimated that 159,000 kg of ARVs may reach the
aquatic environment every year in South Africa (Swanepoel et al.,
2015).
Due to the high prevalence of HIV in South Africa, the importance of
ARV treatment cannot be understated. This treatment has improved the
⁎
quality of life and wellbeing of patients infected with HIV (Bastos et al.,
2016). The high prevalence and treatment of HIV in South Africa render
it likely that there will be a continued increase in the presence of ARVs
in the aquatic environment. This poses a potential risk of aquatic ARV
pollution in South Africa.
Efavirenz is a non-nucleoside reverse transcriptase inhibitor
(NNRTI) which has been widely used in South Africa to treat various
mutant strains of HIV-1 since 1998 and is the third most used ARV
worldwide (Bastos et al., 2016). It is hazardous in the environment as it
is persistent and toxic to aquatic life (Cayman Chemical, 2014;
Stockholm County Council, 2014), and therefore was chosen as the ARV
of interest in this study.
Efavirenz, like other pharmaceuticals, is released into the environment primarily through treated wastewater as waste water treatment
works (WWTWs) are not designed to remove complex compounds such
as pharmaceuticals from effluent. Many pharmaceuticals are only partially removed during water purification (Frederic and Yves, 2014;
Prasse et al., 2010). A previous study reported that efavirenz concentrations in wastewater influent at a South African WWTWs were as
Corresponding author.
E-mail address: inawagenaar@gmail.com (G.M. Wagenaar).
http://dx.doi.org/10.1016/j.etap.2017.09.017
Received 7 April 2017; Received in revised form 13 September 2017; Accepted 20 September 2017
Available online 02 October 2017
1382-6689/ © 2017 Elsevier B.V. All rights reserved.
Environmental Toxicology and Pharmacology 56 (2017) 225–232
L. Robson et al.
2.2. Experimental design
high as 17 400 ng/l and that only 50% of the drug was removed by
wastewater treatment (Schoeman et al., 2015). The amount of efavirenz
removed during the treatment process varies depending on the operation of the WWTW as well as the nature of influent.
Until now, the concentration of efavirenz in surface water in South
Africa was unknown. Wood et al. (2015) reported unquantifiable levels
of efavirenz in surface water in the Roodeplaat Dam system and the
Vaal Dam inflow and outflow. Another study identified efavirenz in the
blood plasma of Clarias gariepinus from the Wasgoedspruit, North West
Province, South Africa at 135 ng/l (Swanepoel et al., 2015).
Literature on the possible effects of efavirenz on fish health at
concentrations found in the aquatic environment is limited. Therefore
the aims of the study were (1) to determine the efavirenz concentration
in water from seven selected sampling sites within the Vhembe District,
South Africa and (2) to determine whether this environmentally-relevant concentration of efavirenz has any effect on the health of
Oreochromis mossambicus. The second aim was achieved by conducting
a 96 h exposure study to efavirenz and establishing the effects on fish
health by using a histology-based fish health assessment index protocol
and various blood parameters and somatic indices.
Sixty five mature O. mossambicus (46% male, 54% female; mean
body mass: 41.64 g ± 1.70; mean total length: 14.70 cm ± 0.17) of
approximately two years of age were selected for the exposure study.
The experiment was conducted in triplicate over a period of three
months (repetition 1: n = 21; repetition 2: n = 20; repetition 3:
n = 24). The fish were allowed to acclimate in a temperature-controlled aquarium for two months prior to the start of the exposure experiment. Thereafter fish were placed into 100 L exposure tanks filled
with municipal tap water where they were acclimated for a further
7 days in a temperature controlled room (28 ± 1 °C; 12:12 light cycle)
prior to exposure. Fish were fed commercial fish pellets daily until
feeding ceased one day prior to exposure.
The fish were divided into four exposure groups including; (1) an
environmentally-relevant efavirenz concentration obtained from the
field study (10.3 ng/l); (2) an efavirenz concentration double that of the
environmentally-relevant concentration (20.6 ng/l); (3) solvent control; and (4) control. Efavirenz was first dissolved in the solvent DMSO
before being added to the exposure tanks. The DMSO used had an assay
of ≥99.5% (gas chromatography) and was selected as the solvent of
choice as it is completely miscible in water. A static testing procedure
was utilised for the exposure study and thus no water exchange regime
was used.
A maximum of two fish were placed into each exposure tank to limit
stress caused by overcrowding, build-up of waste and depletion of
oxygen (Van Dyk et al., 2007). The pH (mean 7.0), TDS (mean
115.4 ppm), EC (mean 229.6 μS/cm), DO2 (mean 6.4 mg/l; 81.4%) and
temperature (mean 28.6 °C) were monitored on a daily basis for each
tank throughout each exposure experiment. Slight variation in parameter values between repetitions were observed. However there was
little variation between exposure groups and all values fell within the
ranges tolerable by Oreochromis spp. (Abubakar et al., 2015). Water
samples for efavirenz measurements were taken at the end of each
exposure experiment.
2. Materials and methods
2.1. Efavirenz analysis
Seven sites in the Vhembe District, South Africa (Fig. 1) were selected for water sampling due to their proximity to anthropogenic activities.
Site 1 was upstream of the local WWTWs in the Mvudi River while
site 2 was below this WWTWs. Site 3 was located at the confluent of the
Mvudi and Dzindi rivers and site 4 at the inflow to the Nandoni Dam.
Site 5 was in the Luvuvhu River where it flows into the Nandoni Dam.
Sites 6 and 7 were in the Dzindi River at points below the main
healthcare facility within the area (Fig. 1).
Water samples were collected from each site in glass bottles and
stored at −4 °C until analysis. Samples were quantitatively analysed for
efavirenz using Liquid Chromatography-Tandem Mass Spectrometry
(LC–MS/MS) at an independent South African National Accreditation
System (SANAS) accredited testing laboratory (Accreditation number
T0410). The efavirenz standard, purchased from Sigma-Aldrich (now
Merck), has a purity of ≥95% (high-performance liquid chromatography) and is soluble in dimethyl sulfoxide (DMSO) (soluble 15 mg/
ml). Physico-chemical water parameters including pH, total dissolved
solids (TDS), electrical conductivity (EC), dissolved oxygen DO2 and
temperature were measured at each site.
2.3. Histology-based fish health assessment
After exposure each fish underwent a necropsy to identify any
macroscopic abnormalities. The necropsy included examination of the
eyes, skin, fins and opercula whereby any abnormality observed was
noted. The fish were then weighed and the total length recorded. These
measurements were used to calculate the condition factor (CF) of each
fish using the following formula (Carlander, 1969):
CF =
Total body mass (g)
x100
Total body length (cm)3
Fig. 1. A map of South Africa showing the Vhembe District circled in red and sampling sites numbered 1–7. (A = position of the WWTWs; B = position of the healthcare facility).
226
Environmental Toxicology and Pharmacology 56 (2017) 225–232
L. Robson et al.
pattern; a = score value; w = importance factor.
Blood was drawn from the dorsal caudal artery at the posterior end
of the fish using a 26 gauge needle and 1 mL syringe and immediately
placed into a lithium heparin vacutainer. The total white blood cell
(WBC) and red blood cell (RBC) counts, differential WBC count, haemoglobin (Hb), haematocrit (Hct) and leukocrit (Lct) were measured
using standard techniques (McCarthy et al., 1973).
Each fish was sacrificed by severing of the spinal cord behind the
head. This procedure was approved by the Ethics Committee of the
Faculty of Science, University of Johannesburg. The liver, gonads,
heart, kidney and second gill arch on the left of each fish were excised
and weighed. The gonads and gill arch were fixated in Bouin’s solution
for 24 h and the heart, kidney and sections of the liver were fixated in
10% neutrally buffered formalin (NBF) for 48 h. The anterior lobe of
the liver and the posterior region of the kidney were sampled for histological assessment. An internal necropsy was done on the gills, bile,
mesenteric fat, liver, spleen, hindgut and kidney.
The recorded weights of the liver, gonads and spleen were used to
calculate the hepato-somatic index (HSI), the gonado-somatic index
(GSI) and the spleno-somatic index (SSI), respectively (Van Dyk, 2006;
Adams et al., 1993) using the following formulae:
HSI =
Liver mass (g)
x 100
Total body mass (g)
GSI =
Gonad mass (g)
x 100
Total body mass (g)
SSI =
Spleen mass (g)
x 100
Total body mass (g)
Ifish =
∑ rp ∑ change
3. Results
3.1. Field study measurements
The highest efavirenz concentration was present at site 1 (10.3 ng/
l), followed by site 7 (5.1 ng/l), site 4 (2.0 ng/l) and site 5 (1.6 ng/l).
Water samples from sites 2, 3 and 6 contained efavirenz concentrations
below the level of quantification (1.0 ng/l). The highest concentration
found at site 1 (10.3 ng/l) was used as the environmentally-relevant
concentration for the exposure study to determine the effect on fish
health. This concentration was doubled (20.6 ng/l) to provide the
second concentration used in the exposure experiment.
3.2. Histology-based fish health assessment
The necropsy indicated macroscopic abnormalities in all groups
except the control group. Four isolated cases of pale gills, skin aberration, sinusoidal congestion and an abnormal fin in separate fish were
observed in the solvent control, 10.3 ng/l exposure group and 20.6 ng/l
exposure group. Other macroscopic abnormalities included focal and
diffuse discolouration of the liver which occurred in all efavirenz exposed groups. The eyes, opercula, spleen, hindgut and kidney of all the
fish were considered normal.
The mean ( ± standard deviation (SD)) CF, somatic indices and
blood parameters for each exposure group are indicated in Table 1. Fish
used for the exposure experiment were in good condition as the mean
and median CF for all groups was above 1 (Van Dyk, 2006; Carlander,
1969). Mean HSI values were low in all groups according to Munshi and
Dutta (1996) (< 1%) however the mean HSI values were similar to
those found in a previous study on O. mossambicus by Van Dyk (2006)
who conducted a study in pollutant-free water to determine baseline
values for O. mossambicus. The GSI and SSI in all groups were similar to
values obtained in unstressed O. mossambicus (Van Dyk, 2006). No
meaningful significant differences were identified between the exposed
groups regarding CF, HSI, GSI, SSI, Hct, Hb, WBC counts and RBC
counts. However, the Lct of the 10.3 ng/l exposure group was significantly higher than that of the 20.6 ng/l exposure group; and the Lct
of the 10.3 ng/l exposure group was significantly higher than that of the
control group (p < 0.02) (Table 1).
Histopathology was identified in the gills, kidney, gonads and heart
however the damage was not significant as no differences (p > 0.02)
were observed in the Iorg for each of these organs. The mean Iorg and Ifish
for each exposure group is shown in Fig. 2. The heart (Iheart) presented
with the least amount of damage and therefore had the smallest mean
Iorg for all groups. The Igills, Ikidney, Iheart and Iovaries were all below 5, and
all organ indices were below 10.
As seen in Fig. 2, the organ with the highest index, and thus the
highest degree of damage, was the liver. The Iliver of the 20.6 ng/l exposure group was significantly higher than the Iliver of the control group
(p < 0.02). No other significant differences were identified between
the Iorg of each group.
Micrographs of the liver damage are displayed in Figs. 3 and 4.
Steatosis of the liver was visible as vacuoles resembling fatty
(aorgrpchange xworprpchange)
where: org = organ; rp = reaction pattern; a = score value; w = importance factor.
Itotrp =
∑ org ∑ change
(aorgrpchange xworprpchange)
where: fish = fish index; org = organ; rp = reaction pattern; a = score
value; w = importance factor.
A high index is representative of a high degree of organ or tissue
damage. The Kruskal-Wallis and Mann-Whitney tests were used to
identify statistical differences between exposure groups. The Bonferroni
correction was applied in order to adjust the significance level to preempt making a Type 1 error and therefore an alpha level of 0.02 was
used when analysing the results (p < 0.02).
Once fixated in Bouin’s solution and 10% NBF, samples were washed overnight ( ± 12 h) in running tap water, dehydrated in increasing
concentrations of ethanol, cleared with xylene and embedded in paraffin wax blocks which were sectioned using a microtome to a thickness
of 5 μm (Bancroft and Gamble, 2008). Samples were then prepared for
histological assessment using standard haematoxylin and eosin (H & E)
staining techniques (Van Dyk, 2006).
Each slide prepared for histological assessment was analysed under
light microscopy (Leica DMLS – ICCA Leica Microsytems, Shangai,
China). Digital micrographs of all histopathology were taken using a
Zeiss Axioplan 2 Imaging system with AxioVision software, version
4.7.2 (12-2008).
A quantitative histological assessment protocol described by Bernet
et al. (1999) and adapted by Van Dyk et al. (2009) was used to quantify
observed histopathology. During histological assessment, any change
observed was given a score value depending on the severity of the
histopathological damage. The degree and extent of the change is described as follows:
0 – No occurrence of histopathological change.
2 – Mild occurrence of histopathological change.
4 – Moderate occurrence of histopathological change.
6 – Severe occurrence of histopathological change.
Histopathology was assigned an importance factor of 1–3 indicating
pathological importance and grouped under seven reaction patterns
including; i. circulatory disturbances; ii. regressive changes; iii. progressive changes; iv. inflammation; v. neoplasms; vi. focal cellular alterations; and vii. intersex. These values were used to calculate an index
for each organ (Iorg), an index for each reaction pattern (Itotrp) and a
total fish index (Ifish) using the following formulae:
Iorg =
∑ org ∑ rp ∑ change
(aorgrpchange xworprpchange)
where: tot rp = total reaction pattern; org = organ; rp = reaction
227
Environmental Toxicology and Pharmacology 56 (2017) 225–232
L. Robson et al.
Table 1
Mean ( ± SD) condition factor (CF), the somatic indices and blood parameters for each efavirenz exposed group.
Control
CF
HSI (%)
SSI (%)
GSI (%)
Hct (%)
Lct (%)
Hb (g/dl)
WBC count
(mm3)
RBC count
(mm3)
Male
Female
Sig.
10.3 ng/l Exposure
Sig.
20.6 ng/l Exposure
Solvent control
20.6 ng/l
Exposure
Sig.
10.3 ng/l
Exposure
20.6 ng/l
Exposure
Control
1.29 ± 0.12
0.57 ± 0.13
0.10 ± 0.09
0.651
0.346
0.042
0.679
0.270
0.138
1.36 ± 0.28
0.59 ± 0.13
0.11 ± 0.06
0.918
0.512
0.558
1.29 ± 0.08
0.62 ± 0.14
0.15 ± 0.09
1.28 ± 0.14
0.72 ± 0.20
0.13 ± 0.08
0.607
0.027
0.220
0.37 ± 0.31
2.41 ± 1.08
0.302
0.424
0.115
0.657
0.31 ± 0.29
2.09 ± 1.42
0.897
1.000
0.21 ± 0.06
2.15 ± 0.74
0.17 ± 0.06
2.15 ± 0.90
0.003
0.728
22.33 ± 5.92
1.59 ± 0.54
6.71 ± 0.98
1985 ± 906
0.547
0.019
1.000
0.105
0.376
0.777
0.176
0.313
23.16 ± 5.19
1.98 ± 0.36
6.69 ± 1.39
2329 ± 1107
0.046
0.011
0.226
0.546
19.73 ± 2.83
1.53 ± 0.49
6.14 ± 1.67
2234 ± 1549
18.8 ± 7.97
1.98 ± 2.07
6.52 ± 1.74
1409 ± 909
0.124
0.852
0.758
0.333
1667044 ± 614645
0.662
0.801
1705271 ± 719645
0.512
1719141 ± 415854
1837140 ± 1168039
0.921
given in Fig. 5. No inflammatory changes or tumours were observed in
any of the fish and thus the II and IT were 0 for all fish. The highest Itotrp
was the regressive change reaction pattern (IRC). This indicates that
regressive changes were the most dominant type of histopathological
change observed throughout all the groups. The IRC of the 10.3 ng/l
exposure group and the 20.6 ng/l exposure group were significantly
higher than that of the control group (p < 0.02) (Fig. 5). No other
significant differences were identified between the Itotrp of the groups.
The Ifish of the 20.6 ng/l exposure group was significantly higher than
that of the control group.
degeneration and was observed in all groups (Fig. 3A). Steatosis foci, as
seen in Fig. 3B, C and D was observed only in efavirenz exposed fish,
whereby fatty degeneration occurred in focal areas as opposed to the
whole liver tissue. On closer inspection (Fig. 3D), the hepatic tissue
surrounding the central vein was atrophied and the presence of nuclear
pleomorphism was evident. Fig. 3E shows the difference between
normal hepatic tissue and necrotic tissue, where the cells have degenerated to form a focal area of necrosis. Fig. 3F illustrates an abnormal abundance of RBCs in the sinusoids and blood vessels indicative
of sinusoidal congestion. Fig. 4A and C display intracellular deposits,
frank necrosis and vacuolation other than steatosis. Large groups of
melanomacrophage centres (MMCs) are shown in Fig. 3B and C and
Fig. 4B, however it has been suggested in literature that these structures
are a normal characteristic in fish tissue (Leknes, 2004).
The total reaction pattern indices (Itotrp) and fish indices (Ifish) are
4. Discussion
A previous exposure study conducted on rats showed that efavirenz
exposure caused an increase in leukocyte infiltration in the mesentery
Fig. 2. The mean organ and fish indices (Iorg and Ifish) for each efavirenz exposed group.
228
Environmental Toxicology and Pharmacology 56 (2017) 225–232
L. Robson et al.
Fig. 3. Histopathological damage to the
liver (haematoxylin and eosin (H & E)
staining) – (A) Portal vein (1) surrounded by
steatosis (2) diffuse throughout liver; (B)
Steatosis foci (3), hepato-pancreatic tissue
(4) and MMCs (5) in the liver; (C) Steatosis
foci (6) surrounding a central vein (7); (D)
Detail of steatosis foci showing nuclear
pleomorphism (8); (E) Necrotic foci (9)
surrounded by normal liver tissue (10); (F)
Sinusoidal congestion (11) within liver
tissue.
2007). We found a significant negative impact on the liver of fish induced by efavirenz exposure. One of the most common damages to the
liver associated with efavirenz treatment is hepatic steatosis. Macias
et al. (2012) described the effect of hepatic steatosis on patients suffering with HIV and hepatitis C and noted an association between
efavirenz treatment and the progression of hepatic steatosis. The researchers observed that the longer a patient received efavirenz treatment, the higher the progression of hepatic steatosis. Another study
reports that liver toxicity is the most relevant adverse effect of ARV
treatment and often leads to clinical hepatitis (Rivero et al., 2007). It
has been established that efavirenz causes mitochondrial toxicity in
hepatocytes and inhibits mitochondrial function inducing bioenergetic
stress in hepatocytes (Blas-García et al., 2010). A reduction in energy
production in the form of adenosine triphosphate (ATP) is mediated by
the enzyme adenosine monophosphate–activated protein kinase
(AMPK) and leads to an accumulation of lipids in the cytoplasm of
hepatocytes causing steatosis (Apostolova et al., 2013; Blas-García
et al., 2010). It was very worrying that steatosis was observed in fish
from all the groups but steatosis foci were observed only in efavirenz
exposed fish. It has been proposed in literature that pharmaceuticals in
(Orden et al., 2014). The study concluded that efavirenz induced interactions between leukocytes and the endothelium, causing an increase in leukocyte movement from blood vessels into tissue as an inflammatory response (Leick et al., 2014). This may explain why fish
exposed to 20.6 ng/l efavirenz presented with significantly lower Lct
values than those exposed to 10.3 ng/l efavirenz. The 20.6 ng/l exposure may have elicited an inflammatory response causing leukocytes
to leave the blood vessels and enter tissue, resulting in a lower Lct.
However, this concept does not account for the significant decrease in
Lct in the control fish compared to 10.3 ng/l efavirenz exposed fish. It
was concluded that this may be due to the fact that the control fish were
less stressed as they were not exposed to efavirenz and therefore presented with fewer leukocytes in the blood and thus a lower Lct (Barton
et al., 2002). Although differences between the Lct of the groups were
identified, all the values were still in the normal range of < 4% as
described by Adams et al. (1993).
Efavirenz is well known to induce hepatotoxic effects in the liver of
humans (Manosuthi et al., 2014; Apostolova et al., 2013; Elsharkawy
et al., 2013; Macias et al., 2012; Pineda et al., 2012; Qayyum et al.,
2012; Yimer et al., 2012; , 2014; Blas-García et al., 2010; Rivero et al.,
229
Environmental Toxicology and Pharmacology 56 (2017) 225–232
L. Robson et al.
Fig. 4. Histopathological damage to the
liver (H & E staining) – (A) Liver tissue with
intracellular deposits (1) and sinusoids (2);
(B) MMCs (3) adjacent to hepato-pancreatic
tissue (4); (C) Necrotic cells (frank necrosis)
(5) and vacuolation other than steatosis (6)
in the liver.
Fig. 5. The mean reaction pattern indices (Itotrp) and fish indices (Ifish) for each efavirenz exposed group.
230
Environmental Toxicology and Pharmacology 56 (2017) 225–232
L. Robson et al.
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the environment may have similar effects on organisms as it has on
humans (Arnold et al., 2013; Kolpin et al., 2002). This statement seems
to be true in relation to the current study as similar liver damage was
observed in fish exposed to efavirenz compared to effects observed in
humans receiving efavirenz treatment.
It is important to note that in human studies, the hepatotoxic effect
of efavirenz was exacerbated in patients who already suffer from liver
problems including cirrhosis (Pineda et al., 2012). Patients are also
increasingly susceptible to hepatotoxic effects by efavirenz if they are
infected with HIV and hepatitis C or tuberculosis (TB) (Yimer et al.,
2014; Pineda et al., 2012). Therefore it can be inferred that efavirenz
exposed fish were susceptible to the toxic effects there-of and may have
developed steatosis foci from an existing condition of steatosis.
However, it is also stated that patients with existing liver problems
are at risk of having their liver problems exacerbated by efavirenz. This
observation can be inferred to the current study whereby fish in the
control group showed microscopic liver damage, whereas fish exposed
to 20.6 ng/l efavirenz had more severe liver damage, as indicated by
the significantly higher Iliver.
Microscopic damage was seen in the gills, liver, kidney, heart and
gonads, and in all the groups. However, the Iliver, IRC and Ifish were
significantly higher (p < 0.02) in the 20.6 ng/l exposure group compared to the control group. The IRC was also significantly higher in the
10.3 ng/l exposure group compared to the control group. This indicates
that the 10.3 and 20.6 ng/l exposure of efavirenz to the fish caused
major liver damage including regressive damage. Regressive damage
includes cellular changes that may result in a decrease in organ function
or total loss of an organ. A significantly higher Ifish in 20.6 ng/l exposed
fish corresponds with an increase in the severity and degree of microscopic damage induced by efavirenz exposure and a decrease in the
overall health of exposed fish.
From this study it is crucial that due to the sheer amounts of ARVs
potentially entering the environment, it is essential to monitor efavirenz
concentrations and identify possible effects thereof on biota. To date,
this is the first study to quantify efavirenz in South African surface
water and to describe the effects of an environmentally-relevant concentration of efavirenz on the health of O. mossambicus.
5. Conclusions
It is clear that 10.3 ng/l efavirenz may severely damage the liver
and lead to lower Lct levels within O. mossambicus (p < 0.02) after
acute exposure. Thus it may be concluded that fish exposed to efavirenz
in the aquatic environment are at risk of developing liver damage, such
as steatosis, caused by efavirenz. Fish with previous liver damage are at
even higher risk as they are more susceptible to the hepatotoxic effects
of the drug. At a concentration of 20.6 ng/l efavirenz has the ability to
impact not only the liver of fish exposed to it, but also the overall fish
health compared to control fish, as described by the Ifish. Regressive
changes were the predominant reaction pattern observed in all fish,
indicating that the histopathological changes caused by acute efavirenz
exposure may eventually lead to functional organ loss and declined fish
health. These results could have serious implications for fish and other
non-target organisms exposed to efavirenz in the aquatic environment.
Acknowledgements
The authors would like to thank the National Research Foundation
of South Africa for financial support (Incentive Funding for Rated
Researchers, UIDs 86056: and Scarce Skills bursary Fund 89871) and
Ms J Van Staden from STATKON at the University of Johannesburg for
her assistance with the statistical analysis.
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