Riset Ilmu Pengetahuan dan Polusi Lingkungan

https://doi.org/10.1007/s11356-018-2839-8

ARTIKEL PENELITIAN

Perilaku dan histopatologi sebagai penanda biologis untuk

evaluasi efek parasetamol dan propranolol pada spesies
ikan neotropis Phalloceros harpagos
Gregorio Nolazco Matus 1 & Beatriz VR Pereira1 & Elaine CM Silva-Zacarin1,2 & Monica Jones
Costa1,2 & André Cordeiro Alves dos Santos2 & Bruno Nunes3,4

Diterima: 16 Februari 2018 / Diterima: 23 Juli 2018

# Springer-Verlag GmbH Jerman, bagian dari Springer Nature 2018

Abstrak
Obat-obatan farmasi di lingkungan air dapat menyebabkan efek merugikan pada organisme non-target.
Penelitian ini bertujuan untuk menilai efek jangka pendek konsentrasi subletal paracetamol dan propranolol
pada ikan Phalloceros harpagos, khususnya preferensi terang / gelap, pola renang, pigmentasi kulit,
histopatologi, dan kadar glikogen hati. Ikan secara akut terpapar pada konsentrasi subletal dari kedua
parasetamol (0,008, 0,08, 0,8, 8, 80 mg L- 1) dan propranolol (0,0001, 0,001, 0,01, 0,1, 1 mg L- 1) dalam
kondisi terkontrol. Untuk skototaksis, preferensi yang signifikan untuk kompartemen gelap diamati untuk
kelompok yang terpapar parasetamol dengan konsentrasi tertinggi (80 mg L- 1). Paparan propranolol secara
signifikan mengubah pola berenang, terutama pada ikan yang terpapar pada0,001 mg L- 1 konsentrasi.
Pigmentasi berkurang pada ikan yang terpapar propranolol (0,1, 1 mg L- 1). Konsentrasi propranolol terendah
(0,0001 mg L−1) menyebabkan penurunan reaksi histokimia glikogen hati. Data ini menunjukkan bahwa obat-
obatan dapat menimbulkan efek subletal pada organisme non-target, bahkan pada konsentrasi rendah,
membahayakan fungsi spesifik individu dengan relevansi ekologis, seperti keseimbangan energi dan
perilaku.

. . . . . .
Kata kunci Guaru Farmasi Daerah tropis Air tawar Histopatologi hati Glikogen hati Toksisitas akut

Pendahuluan
Editor yang bertanggung jawab: Cinta Porte
Materi tambahan elektronik Versi online artikel ini Peningkatan populasi manusia global dan
(https://doi.org/10.1007/s11356-018-2839-8) berisi materi perkembangan proses industri, serta pertumbuhan
tambahan, yang tersedia untuk pengguna yang berwenang.
penggunaan bahan yang berbeda dalam proses
produksinya, telah menimbulkan dampak terhadap
* Bruno Nunes
nunes.b@ua.pt
lingkungan, termasuk pencemaran atmosfer, tanah,
dan sistem akuatik (Moiseenko 2008). Kontaminasi
1
Pós-Graduação em Biotecnologia e Monitoramento badan air dapat terjadi karena pelepasan bahan
Ambiental (PPGBMA), Universidade Federal de São kimia yang berasal dan keanekaragaman alam di
Carlos (UFSCar), Kampus Sorocaba, Rodovia João Leme lingkungan, seperti pestisida, surfaktan,
dos Santos km 110, Itinga, Sorocaba, SP 18052- 780, hidrokarbon, dibenzofuran poliklorinasi, logam (van
Brasil
der Oost et al. 2003), dan obat-obatan farmasi untuk
2
Departamento de Biologia, Universidade Federal de São keduanya penggunaan manusia dan dokter hewan
Carlos, Rodovia João Leme dos Santos km 110,
Itinga,
(Rodrigues et al. 2014; Fabbri 2015; Fabbri dan
Sorocaba, SP 18052-780, Brasil Franzellitti 2016).
3
Departamento de Biologia, Universidade de Aveiro,
Obat farmasi adalah senyawa kimia yang
Campus de Santiago, 3810-193 Aveiro, Portugal memiliki banyak karakteristik unik yang membuatnya
4 sangat relevan dengan lingkungan karena dapat
Centro de Estudos do Ambiente e do Mar (CESAM,
Laboratório Associado), Universidade de Aveiro, tahan terhadap degradasi metabolik. Dengan cara
Campus de Santiago, 3810-193 Aveiro, Portugal ini, setelah penggunaan terapeutiknya, obat-obatan
biasanya dikeluarkan pada akhir penggunaannya
oleh manusia dan / atau hewan, hadir baik dalam

bentuk yang tidak berubah (senyawa induk) atau

sebagai metabolit (Cleuvers 2003; Daughton dan
Ternes 1999) di menerima perairan (Ternes pada kadar 10 μg L−1 (Fent et al. 2006). Baru-baru
1998;Miao et al 2002;.Sodré et al 2010;..Ahmed et ini, 17 μg L−1 tingkat
al 2017).Akibatnya, obat-obatan bersifat pseudo-
persisten karena emisi berkelanjutannya dari
instalasi pengolahan limbah sama dengan tingkat
pembuangannya ke lingkungan akuatik (Petrović et
al. 2003). Obat aktif secara biologis dan dapat hidup Singapura, tingkat maksimum parasetamol adalah
berdampingan dan berinteraksi dengan obat lain di
lingkungan, menyebabkan efek sinergis atau
antagonis (Cleuvers 2003). Selain itu, obat
merupakan zat lipofilik sedang, suatu faktor yang
memungkinkan obat melewati membran biologis,
sehingga lebih mudah diserap oleh organisme
(Halling-Sorensen et al. 1998). Dengan demikian,
obat merupakan senyawa yang berpotensi
membahayakan biotain lingkungan (Nunes et al.
2005; Owen et al. 2007). Zat-zat ini telah terdeteksi stres oksidatif, sebuah fenomena yang telah
dalam kisaran nanogram per liter hingga mikrogram
per liter (Schrap et al. 2003) dan hingga miligram
per liter di ekosistem perairan (Nunes et al. 2014). 2015.).Parasetamol
Beberapa penelitian telah menunjukkan bahwa
obat-obatan, dengan konsentrasi serendah kolinesterase tidak langsung melalui oksidasi enzim
nanogram per liter, mampu menyebabkan efek
biologis yang merusak pada biota perairan (Schrap
et al. 2003; Nunes et al. 2005; Franzellitti et al.
2011; Fabbri 2015; Fabbri dan Franzellitti 2016),
pada tingkat molekuler, seluler, dan individu. Pada
tingkat sel, histopatologi hati ikan merupakan
biomarker yang baik untuk menilai pencemaran
perairan, karena organ ini memetabolisme senyawa
kimia, seperti farmaseutikal, yang dapat memicu
perubahan histologis hati yang merupakan respons
terhadap adanya sublethal stressor (Bernet et al.
1999). Di antara perubahan pada tingkat individu,
substansi tersebut juga dapat mengubah sifat
perilaku organisme air (Van Donk et al. 2016),
termasuk ikan.
Di antara beberapa obat yang dapat ditemukan di
lingkungan, adalah mungkin untuk menyoroti
parasetamol, juga dikenal sebagai asetaminofen
atau N-asetil-p-aminofenol (APAP), yang
merupakan obat analgesik dan antipiretik yang
banyak digunakan dalam terapi manusia ( Tunggal
et al. 2010).Meskipun secara umum dianggap
sebagai obat yang aman dalam dosis terapeutik,
parasetamol sangat toksik bagi manusia yang
mengalami overdosis (Wallace 2004; Jozwiak-
Bebenista dan Nowak 2014). Di lingkungan akuatik,
parasetamol telah dianggap sebagai obat dengan
persistensi tinggi (de Voogt et al. 2009), dengan
waktu paruh sekitar 2 minggu (Loffler et al. 2005).
Oleh karena itu, sangat penting untuk mengevaluasi
efek potensinya pada organisme bukan target
(Ramos dkk. 2014) karena dapat menjadi racun
bagi organisme akuatik (Heberer 2002; Nunes dkk.
2014; Nunes dkk. 2015).
Obat ini sering terdeteksi di lingkungan akuatik penghambatan cat echolamines (CAs) yang
dalam tingkat yang cukup (Chaouchi dan Hamdaoui
2014) seperti 65 μg L−1 di Sungai Tyne di Inggris
(Roberts dan Thomas 2006) dan di atas 78 μg L −1 di
Sungai Danube ( Grujić et al. 2009).Di stasiun
pengolahan limbah di Eropa (Ternes 1998),
parasetamol ditemukan dalam kadar 6 μg L- 1, dan di
AS, parasetamol dikuantifikasi dalam limbah sungai
Environ Sci Pollut Res
parasetamolditemukan di limbah cair di Australia
utara (French et al. 2015), dan di reservoir air di
485,5 ng L−1 (Anda dkk. 2015). Studi di muara
Sungai Parramatta di Australia juga menunjukkan
adanya parasetamol pada level 0,1 ng L- 1 (Birch et
al. 2015). Di Brasil, kadar parasetamol yang diukur
adalah 30,421 ng L−1 (Campanha et al. 2015) di
Sungai Monjolinho di São Carlos, Negara Bagian
São Paulo.
Secara umum, hasil toksisitas parasetamol dari
dijelaskan untuk spesies akuatik eral sev (Xu et al
2008;.Ramos et al 2014;.Nunes et al
adalah senyawa pro-oksidatif
yang dapat memberikan efek penghambatan
asetilkolinesterase (AChE) (Nunes 2011; Nunes et
al. 2015), sehingga mempengaruhi perilaku alami
ikan (Nunes 2011; Nunes et al. al. 2015). Selain itu,
ada penelitian tentang efek jangka pendek histologis
paraceta mol pada tikus (Merrick et al 2006.)Dan
tikus (Li et al 2013;.Alamgeer et al
2017;..Papackova et al 2018),serta di ikan zebra
(North et al. 2010) terpapar konsentrasi lebih rendah
daripada yang diuji pada hewan pengerat ini. Dalam
skenario ini, hipotesis kami adalah bahwa
parasetamol dapat mengubah perilaku dan jaringan
hati ikan neotropis.
Obat lain yang sering ditemukan di lingkungan
perairan adalah propranolol (Cleuvers 2005; Godoy
et al. 2015), yang merupakan obat terapeutik yang
banyak digunakan dalam pengobatan penyakit
jantung manusia (Al-majed et al. 2017). Keberadaan
obat ini ditentukan di Inggris pada air limbah (desa
Cilfynydd), dimana konsentrasinya berkisar antara
16 hingga 388 ng L-1 (Zhou et al. 2009), dan juga di
perairan permukaan, meskipun pada konsentrasi
yang lebih rendah, berkisar antara 10 sampai 215
ng L-1 (Kim et al. 2009), dalam limbah cair di Inggris
(0,06-0,199 dan 0,093 μg L-1; Roberts dan Thomas
2006; Ashton et al. 2004). Propranolol juga dihitung
dalam limbah rumah sakit di Taiwan, dan tingkat
yang diukur mencapai 0,225 μg L- 1 (Lin dan Tsai
2009), dan juga ditemukan di Prancis, dalam limbah
dari instalasi pengolahan air limbah (0,01– 0,04 dan
0,089 μg L−1; . Coetsier et al 2009;Andreozzi et al
2003)..Senyawa ini memiliki perkiraan waktu paruh
5,7 hingga 28,5 hari (Yin et al. 2017).
Propranolol adalah penghambat non selektif dari
reseptor β-adrenergik, yang efeknya dimediasi oleh
mengikat β-adrenoreseptor (Gether 2000). β-
Adrenoreseptor adalah protein reseptor
transmembran yang digabungkan dengan protein G
dalam membran plasma sel, yang, setelah aktivasi,
meningkatkan sintesis cAMP. cAMP adalah
pembawa pesan sekunder dari jalur transduksi
sinyal yang terlibat dalam proses fisiologis yang
 berbeda, seperti transmisi saraf, pertumbuhan, ekotoksikologi bahan kimia yang diduga di
metabolisme, diferensiasi sel, sekretifikasi, dan lingkungan akuatik, terutama yang disesuaikan
pertahanan imunologi (Hoelz et al. 2013). Dalam dengan kondisi tropis.
kasus reseptor β-adrenergik, jalur pensinyalan
berpasangan protein G bekerja padafisiologis jangka pendek konsentrasi subletal paracetamol
antihipertensi
Reseptor Polut Lingkungan Sci
proses, dan oleh karena itu, kelas protein reseptor kulit, histopatologi, dan kadar glikogen di hati.
ini telah menjadi target terapi obat (Hoelz et al.
2013). Propranolol juga bertindak sebagai antagonis
serotonin (Alexander dan Wood 1987; Huggett et al.
2002; Stanley et al. 2006). Karena sifat-sifat ini
sebagai penghambat adrenoreseptor β, propranolol
berpotensi menyebabkan perubahan penting pada
spesies ikan, yaitu penurunan pigmen kulit
(Miyashita dan Fujii 1975; Sköld et al. 2016). Selain
itu, obat ini dapat mempengaruhi mobilisasi glikogen
hati (Kotoulas et al 2006;.Fabbri dan Bulan
2016),yang memainkan peran kunci dalam
mengatur asupan makanan dan glukosa darah
penyimpanan energi homeostasis. Dengan
mengubah keseimbangan energi, propranolol dapat
mempengaruhi berenang perfor Mance pada ikan
(Crespel et al. 2017).Dalam konteks ini, hipotesis
kami adalah bahwa propranolol dapat mengubah
perilaku dan menentukan kadar glikogen pada
spesies ikan neotropis, yaitu Phalloceros harpagos.
Semua uji ekotoksikologi berbasis ikan modern
dilakukan mengikuti pedoman standar yang
dilakukan dengan spesies secara eksklusif dari
zona beriklim sedang (misalnya, Pedoman OECD
(Organisasi untuk Kerjasama Ekonomi dan
Pembangunan) untuk Pengujian Bahan Kimia
1992a, b, 1998, 2000). Oleh karena itu, perlu dibuat
standar baru untuk pengujian ekotoksikologi dengan
spesies ikan lain, yaitu ikan yang berasal dari
neotropis, yang akan lebih mewakili sistem akuatik
di wilayah dunia tersebut. Spesies neotropis ini,
dengan sepenuhnya beradaptasi dengan kondisi
abiotik tropis, cenderung merespons secara lebih
realistis dan, dengan relevansi ekologis yang
meningkat, terhadap kondisi yang ditemukan di
lingkungan tempat mereka tinggal. Standarisasi uji
coba ekotoksikologi dengan spesies neotropis
mungkin, di masa depan, dapat menjadi faktor kunci
dalam program pemantauan di negara tropis,
seperti Brasil.
Ikan air tawar neotropis Phalloceros harpagos
(Lucinda 2008; nama umum: guaru)
(Cyprinodontiformes: Poeciliidae) memiliki potensi
besar untuk digunakan dalam uji ekotoksikologi.
Spesies ini dipilih sebagai organisme uji karena
merupakan ikan asli dari cekungan sungai Paraná
dan Paraguay (Lucinda 2008), sehingga sangat
mewakili ekosistem tropis air tawar. P. harpagos
adalah konsumen sekunder, menampilkan
dimorfisme seksual, dan mudah ditangkap,
berukuran kecil, dan mudah beradaptasi dengan
kondisi laboratorium dalam pemeliharaan dan
pengujian. Karakteristik ini mengubah P. harpagos
menjadi organisme yang ideal untuk pengembangan
pengujian berbasis laboratorium untuk penilaian
Penelitian ini bertujuan untuk mengevaluasi efek
dan propran olol pada parameter perilaku ikan
Phalloceros harpagos, khususnya preferensi
terang / gelap (scototaxis), pola renang, pigmentasi
Bahan dan metode
Bahan kimia
Asetaminofen (nomor CAS 103-90-2 / uji 98-102%) dan
propranolol (nomor CAS 318-98-9 / uji ≥ 99%) dibeli
dari Sigma-Aldrich ™ St. Louis, MO, AMERIKA
SERIKAT.
Koleksi
Individu P. harpagos dikumpulkan di sungai di
daerah pedesaan kotamadya Cabreúva (Negara
Bagian São Paulo, wilayah tenggara Brasil, 23 ° 18 ′
23.8 ″ S, 47 ° 05 ′ 50.4 ″ W), yang terletak di batas-
batas Serra do Japi.
Pengumpulan ikan disahkan oleh CEUA (Komite
Etik Penggunaan Hewan dari Universitas Federal
São Carlos) dan SisBio (Sistem Otorisasi dan
Informasi dalam Keanekaragaman Hayati); 180 ikan
(n = 180), jantan dan betina yang belum dewasa
(dengan ukuran 2 ± 0,5 cm), dikumpulkan secara
manual dengan jaring bermata sempit dan
ditempatkan di kotak khusus untuk mengangkut
ikan, dengan pompa udara untuk mengoksidasi air. .
Pengangkutan dan aklimasi organisme uji
Setelah pengumpulan, ikan diangkut ke Universitas
Federal São Carlos, Kampus Sorocaba. Ikan
diaklimatisasi selama 12 hari dalam akuarium kaca
dan selama 5 hari dalam pot plastik yang digunakan
untuk bioassay sebelum memulai paparan, di
bawah kondisi abiotik yang terkontrol (langkah
terakhir ini wajib untuk mengurangi stres yang
melekat pada prosedur paparan). Selama kedua
periode aklimasi, ikan disimpan dalam air untuk
konsumsi manusia (Klarina®), pada suhu 23 ° C (±
1 ° C) dan fotoperiode 12 jam terang / 12 jam gelap.
Selama periode aklimasi, parameter air dipantau
setiap hari (pH, konsentrasi amunisi, kekerasan
total, kekerasan karbonat, konsentrasi nitrit dan
nitrat). Hewan diberi makan ad libitum dengan
pakan ikan flokulasi untuk ikan tropis (Tetra Min®)
dan dipelihara di akuarium 43-L, dengan inspeksi
harian untuk menjaga kualitas air di akuarium dan
kesehatan organisme.
Paparan akut parasetamol dan propranolol
Tes dilakukan sesuai dengan Guideline Test No.
 203: ikan, uji toksisitas akut (OECD 2009), dengan Scototaxis Tes
modifikasi berikut: hewan secara individual
dipaparkan dalam pot plastik yang digunakan untuk ini dilakukan 72 jam setelah dimulainya eksposur,
konsumsi air oleh manusia, yang sebelumnya dicuci seperti yang dijelaskan oleh Rodrigues et al. (2012)
dengan air deionisasi dan dikeringkan. Tiap pot dan Brandão et al. (2013), diadaptasi dari Maximino
plastik berisi ikan dengan volume 250 mL air et al. (2010). Untuk menghindari hewan terkena
mineral yang digunakan untuk konsumsi manusia stres tambahan pada hari eutanasia (96 jam setelah
(Klarina®), mengikuti proporsi 1 g ikan dalam 1 L dimulainya paparan), penilaian perilaku ini dilakukan
air, seperti yang dijelaskan dalam standar OECD pada hari sebelumnya (72 jam setelah dimulainya
yang diadopsi. Larutan stok parasetamol dan paparan).
propranolol juga disiapkan dengan air mineral Lima ikan yang dipilih secara acak digunakan per
untukmanusia kelompok, dan perilaku masing-masing hewan
dievaluasi secara individual.
Polut Lingkungan Sci
konsumsi(Klarina®). Semua tes termasuk kelompok
kontrol (hewan hanya terpapar air tanpa obat- Hewandikeluarkan dari pot plastik (di mana mereka
obatan). Konsentrasi yang digunakan untuk akut terkena obat) dan segera direndam dalam akuarium
didasarkan pada LC50 data untuk organisme yang yang dimodifikasi untuk mengevaluasi perilaku
sama (mengingat tidak adanya data untuk harpagos individu mereka. Selanjutnya, ikan yang sudah
P. dalam literatur) dan data studi pemantauan diserahkan ke behavioral asessment dikembalikan
lingkungan untuk obat ini. LC50 nilai-nilai yang ke pot paparan untuk melanjutkan paparan obat. Uji
diadopsi sebagai batas atas dari konsentrasi skototaksis dilakukan mulai pukul 08.00 hingga
berkisar, dan tingkat sudah dilaporkan terjadi untuk 12.00. Uji skototaksis dilakukan di akuarium
lingkungan air adalah batas bawah dari kisaran ini. berukuran 18 L, panjang 40 cm x lebar 19 cm x 24
Jadi, untuk parasetamol expo akut yakin, cm, yang dibagi menjadi tiga kompartemen dengan
konsentrasi obat didasarkan pada kedua (i) LC 50 dari area berbeda. warna (putih / netral / hitam), dengan
800 mg L-1,nilai dihitung untuk ikan medaka (Oryzias ukuran 14 cm gelap × 12 cm netral × 14 cm putih.
latipes) (Yamamoto et al. 2007),serta seperti dalam Tiga kompartemen di akuarium dipisahkan secara
(ii) konsentrasi yang telah dilaporkan di lingkungan. fisik. Media yang digunakan untuk analisis ini adalah
Prosedur yang sama diikuti untuk akut untuk air mineral (Klarina®) juga digunakan untuk
propranolol, denganLC50 nilaidari 11,40 mg L-1 untuk karantina / pajanan tetapi tanpa obat. Level medium
latipes O. (Kim et al. 2009).Konsentrasi yang di akuarium ini adalah 10 cm.
ditemukan di lingkungan (batas bawah kisaran Awal pengujian terjadi dengan penempatan ikan
konsentrasi yang diadopsi) adalah 0,8 μg L−1 untuk di kompartemen netral akuarium, di mana mereka
parasetamol (Sodré et al. 2007) dan 0,015 μg L−1 tinggal dari 15 hingga 20 detik untuk aklimatisasi,
untuk propran olol (Campanha et al. 2015). Hewan sebelum melepas pemisah. Periode ini cukup lama
dengan demikian diekspos secara akut ke lima untuk memungkinkan aklimatisasi ikan saat
konsentrasi dari masing-masing obat: 0,008, 0,08, dipindahkan ke akuarium, tetapi cukup singkat untuk
0,8, 8, dan 80 mg L-1 parasetamol dan 0,0001, mencegah kemungkinan pemulihan ikan setelah
terpapar obat (Brandão et al. 2013). Setelah periode
0,001, 0,01, 0,1, dan 1 mg L-1 propranolol. Eksposur
15-20 detik ini, pemisah secara perlahan dilepas
akut dilakukan selama 96 jam dalam sistem
untuk mengevaluasi preferensi ikan untuk
semistatis (perubahan air dan penggantian
konsentrasi obat setiap 48 jam). Lima belas ikan kompartemen hitam atau putih. Ikan tetap berada di
digunakan per konsentrasi, setiap ikan merupakan dalam akuarium selama 5 menit (300 detik), seperti
replika individual, dengan total 90 hewan untuk yang dijelaskan oleh Rodrigues et al. (2012) dan
setiap paparan. Brandão et al. (2013), di mana perpindahan hewan
dievaluasi.
Penentuan biomarker Perilaku ikan dipantau segera setelah pemisah
dilepas, dan selama periode ini, perilaku mereka
direkam dengan menggunakan kamera video digital
Setelah 96 jam pemaparan, ikan di-eutanasia
resolusi tinggi (Model: Sony Handycam DCR-SR47),
dengan hipotermia, yaitu perendaman dalam buffer
yang dipasang 50 cm di atas akuarium.
fosfat sedingin es sampai mereka kehilangan
Pencahayaan buatan juga ditempatkan di atas
kemampuan berenang dan kehilangan refleks
akuarium untuk mencegah hewan mengamati
meluruskan dan gerakan operkuler, diikuti dengan
keberadaan manusia dan untuk memastikan bahwa
pemenggalan kepala. Prosedur ini terbukti cepat,
ikan memiliki perilaku yang paling alami. Video
efektif, dan tidak terlalu menyusahkan dan tidak
tersebut kemudian dianalisis untuk menghitung
menunjukkan bukti kelainan histologis (Wilson et al.
persentase waktu selama ikan berada dalam
2009). Sebelum euthanasia, tes perilaku dilakukan
keadaan gelap dan di kompartemen bening; setelah
pada momen bioassay yang berbeda. Setelah
pengujian ini, ikan dengan hati-hati dikeluarkan dari
pengorbanan, ikan dibedah untuk mengisolasi hati
akuarium dengan jaring dan dikembalikan ke pot
yang digunakan untuk analisis histologis.
plastik masing-masing, untuk melanjutkan paparan
kontaminan sampai selesai 96 jam. Parameter
Tes Perilaku Tes
 berikut diukur: persentase waktu di kompartemen (paraformaldehyde 4% buffered dalam 0,1 M PBS,
hitam, persentase waktu di kompartemen netral, dan pH 7,4). Setelah 48 jam fiksasi pada suhu 4 ° C,
persentase waktu di kompartemen putih; organ dicuci dalam buffer PBS (0,1 M, pH 7,4) dan
pengukuran ini tidak bergantung satu sama lain, dan dimasukkan ke dehidrasi lambat dalam rangkaian
diperoleh setelah analisis visual dari video yang larutan etanol dingin (15, 30, 50, 70, 85, 90, 95,
direkam. 100% — untuk jangka waktu 1 jam setiap mandi),
sesuai dengan protokol yang dijelaskan oleh Silva-
Berenang Zacarin et al. (2012).
Setelah dehidrasi, hati awalnya direndam dalam
Dalam penelitian ini, kami juga mengevaluasi larutan resin cair (Technovit® 7100) + 100% etanol
perilaku (posisi dan pergerakan di kolom air) ikan, (1: 1) selama 2 jam pada suhu 4 ° C, dan
sebagai indikator efek toksik yang diinduksi oleh selanjutnya, hati direndam dalam resin cair murni
obat, parasetamol dan propranolol. Untuk evaluasi selama 48 jam pada suhu 4 ° C. Secara berurutan,
pergerakan dan penempatan ikan di kolom air, hati dipindahkan ke cetakan plastik dan ditanamkan
klasifikasi diadopsi pada resin cair yang mengandung pengeras.
Environ Sci Pollut Res Polimerisasi resin terjadi di dalam kompor pada
suhu 45 ° C selama 2 jam. Sampel yang
dipolimerisasi dikeluarkan dari cetakan alas em dan
menjadi dua jenis perilaku, perilaku berenang khas
dipotong menjadi ketebalan 3 μm dengan
dan atipikal, yang dipantau setiap hari pada interval
waktu yang ditentukan (8, 13, 18 h) selama
eksposur. Ming renang yang khas dipertimbangkan
ketika ikan tetap berenang di dasar pot plastik mikrotom (Leica, RM 2255). Bagian histologis
selama sebagian besar waktu (horizontal) atau dikumpulkan dalam water bath pada suhu 37 ° C
ketika ikan berenang dari atas ke bawah (atau dan ditempatkan pada slide kaca yang telah
sebaliknya) dalam bentuk kontinu (vertikal). dibersihkan sebelumnya.
Perenang atipikal diperhitungkan saat ikan berenang Untuk analisis histologis, slide yang berisi bagian
di bagian atas kapal (horizontal di atas) atau saat hati ikan diwarnai dengan hematoxylin (30 menit)
ikan tidak ada aktivitas berenang, tetap dalam posisi dan eosin (10 menit) -HE, menurut Junqueira dan
diam di dasar. Ikan secara individual dan langsung Junqueira (1983). Untuk analisis histokimia glikogen,
diamati selama 10 menit (n = 15 replikat per yang dideteksi dengan reaksi PAS (asam periodik
kelompok eksperimen), dan data yang dikumpulkan Schiff), slide yang berisi bagian histologis direndam
setelah 96 jam terpapar obat kemudian diubah dalam asam periodik (30 menit) dan, selanjutnya,
menjadi persentase (%) dari jumlah total individu per dalam reagen Schiff (1 jam). Setelah periode ini,
percobaan tal. kelompok untuk analisis statistik. slide dicuci dalam air mengalir selama 30 menit
untuk bukti reaksi positif dalam warna magenta
Pigmentasi (merah muda tua), yang mengindikasikan glikogen
(Mcmanus 1946).
Selain itu, beberapa bagian histologis hati
Ikan mengubah pola pigmentasi tergantung pada
dimasukkan ke pewarnaan xylidine Ponceau untuk
rangsangan lingkungan (termasuk kontaminan);
deteksi protein (Vidal 1970), serta Sudan Black
Oleh karena itu, evaluasi karakteristik ini digunakan
untuk deteksi lipid (Pearse 1960). Hal ini dilakukan
sebagai titik akhir untuk mengevaluasi efek dari
untuk membandingkan dengan data yang diperoleh
kedua obat, parasetamol dan proprano lol. Catatan
untuk reaksi PAS di ma parenkim hati, yang
observasi harian langsung dari 15 ikan per
bertujuan untuk mengkarakterisasi konten yang
kelompok percobaan didaftarkan dan dipantau pada
disimpan di dalam vakuola di hepatosit dari bagian
interval yang ditentukan (8, 13, 18 jam) selama
hati di mana terdapat vakuolisasi.
pemaparan. Pigmentasi diklasifikasikan menjadi dua
kategori: pigmentasi (daerah punggung-ekor sedang Dari setiap ikan per kelompok eksperimen (n =
dan gelap) dan nonpigmentasi (warna terang daerah 5), 2 slide dianalisis per individu, menjadi 3 bagian
punggung-ekor). Setiap kategori dinyatakan sebagai histologis nonsequential dianalisis di setiap slide,
persentase (%) dari jumlah total individu per total 6 bagian hati per individu dari setiap kelompok
kelompok eksperimen (n = 15 ulangan per kelompok eksperimen, yaitu, kelompok kontrol dan hewan
eksperimen). yang terpapar berbeda konsentrasi parasetamol dan
propranolol. Analisis kualitatif dan semikuantitatif
selanjutnya dilakukan di fotomikroskop bidang
Analisis histopatologi cahaya (Leica, DM1000). Analisis dilakukan dengan
menggunakan perangkat lunak Leica Application
Histologi dan histokimia Suite V3.8 (LAS V3.8).

Lima ikan dari masing-masing kelompok Analisis semikuantitatif data histologis

eksperimen disuntik mati (lihat prosedur di atas,
bagian BPenentuan penanda biologis^). Hati dari
Deskripsi dan evaluasi perubahan histologis
lima ikan per kelompok eksperimen (n = 5) diisolasi
dilakukan sesuai dengan protokol Bernet et al.
dan direndam dalam larutan fiksatif
(1999) untuk ikan. Dua parameter ditetapkan untuk
 menentukan indeks perubahan hati individu dari kuantitatif pada awalnya diserahkan untuk uji
masing-masing kelompok eksperimen, yaitu skor (α) normalitas Shapiro Wilk. Hasilnya dibandingkan
dan faktor kepentingan (w). Pengamatan antara kelompok kontrol dan kelompok yang
histopatologi diklasifikasikan dalam skor mulai dari 0 terpapar obat, menggunakan analisis nonparametrik
sampai 6 tergantung pada derajat dan luasnya Kruskal-Wallis. Perangkat lunak statistik yang
perubahan: 0 — tidak ada kejadian, 2 — kejadian digunakan adalah GraphPad Prism 6.
kecil, 4 — kejadian sedang, dan 6 — kejadian
parah. Faktor kepentingan ditetapkan untuk setiap
lesi, sesuai dengan kepentingan patologisnya Hasil
(karena dapat mempengaruhi fungsi organ dan
kelangsungan hidup ikan). Perubahan Tes Perilaku Tes
diklasifikasikan menjadi tiga faktor: (1) kepentingan
patologis minimal (kerusakan dapat dengan mudah
dipulihkan), (2) kepentingan patologis sedang Scototaxis — parasetamol
(kerusakan dapat dipulihkan dalam banyak kasus),
dan (3) kepentingan patologis yang parah Pajanan parasetamol bertanggung jawab atas
( kerusakan tidak dapat diperbaiki, menyebabkan perbedaan yang signifikan antara hewan dari
hilangnya sebagian atau total fungsi organ). Dengan kelompok kontrol dan mereka yang terpapar 1
dengan
mengalikan skor dengan faktor kepentingan setiap konsentrasi tertinggi (80 mg L- ), dengan
perubahan, indeks perubahan untuk setiap ikan peningkatan yang signifikan dalam preferensi ikan
untuk kompartemen gelap (df = 5.24; F = 3.129; p =
0.0258) (Gbr. 1a).
Res Polut Lingkungan Sci
dihitung menggunakan persamaan berikut: Indeks ind
= Σ ( 푤 × α). Dari indeks individu, nilai rata-rata dan
deviasi standar (SD) dihitung untuk semua
kelompok eksperimen.

Analisishasil histokimia

semikuantitatif dariData dari analisis semikuantitatif

histokimia untuk deteksi gly cogen (PAS)
diklasifikasikan menurut intensitas pelabelan PAS-
positif: 0 — tidak adanya reaksi positif, 1-intensitas
reaksi positif yang lemah, 2-intensitas rata-rata
reaksi positif, 3 — intensitas reaksi positif yang kuat,
dan 4 — intensitas reaksi positif yang sangat kuat.
Nilai rata-rata dan SD dihitung untuk semua
kelompok eksperimen.

Analisis statistik

Biomarker perilaku

Untuk evaluasi pola renang dan pigmentasi,

dilakukan analisis chi-square (analisis data non-
kategori) yang membandingkan kelompok kontrol
dengan konsentrasi obat yang berbeda.
Untuk pengamatan scototaxis, data perilaku (%
dari waktu dalam kompartemen gelap) sebelumnya
diserahkan ke formasi trans busur, dan analisis
varian searah (ANOVA satu arah) diikuti oleh uji
multikomparasi Dunnett (dalam kasus ANOVA yang
Perbedaan) kemudian dilakukan untuk mengamati
perbedaan yang signifikan antara konsentrasi obat
yang bervariasi dan kelompok kontrolnya masing-
masing. Tingkat signifikansi yang diadopsi adalah
0,05. Gbr. 1 a Hasil yang diperoleh untuk uji scototaxis —
parasetamol menunjukkan waktu permanen ikan di
kompartemen gelap; nilai sesuai dengan rata-rata 5 hewan
Biomarker sel dengan kesalahan standar masing-masing, kelompok kontrol
dan 5 konsentrasi parasetamol yang berbeda. b Pola
berenang diklasifikasikan dalam renang biasa vs. renang
Data dari analisis histopatologi dan histokimia semi atipikal; nilai sesuai dengan rata-rata 14 hewan. c Jumlah
hewan dengan pigmentasi setelah terpapar parasetamol,
 diklasifikasikan sebagai berpigmen dan tidak berpigmen; nilai
sesuai dengan rata-rata 14 hewan. * - perbedaan signifikan
dalam kaitannya dengan kelompok kontrol,p <0,05
ujiScototaxis - propranolol
Analisis statistik menunjukkan bahwa propranolol
tidak menyebabkan perbedaan yang signifikan
antara hewan dari kelompok kontrol dan mereka
yang terpapar obat yang berbeda konsentrasi (df =
5,24; F = 0,625; p = 0,683) (Gbr. 2a).
Res Polut Lingkungan Sci
Gbr. 2 a Hasil yang diperoleh untuk uji scototaxis —
propranolol menunjukkan waktu permanennya ikan di
kompartemen gelap; nilai sesuai dengan rata-rata 5 hewan
dengan kesalahan standar masing-masing, kelompok kontrol
dan 5 konsentrasi propranolol yang berbeda. b Pola berenang
diklasifikasikan dalam renang biasa vs. renang atipikal; nilai
sesuai dengan rata-rata 14 hewan. c Jumlah hewan dengan
pigmentasi setelah terpapar propranolol, diklasifikasikan
sebagai berpigmen dan nonpigmentasi; values correspond to
the average of 14 animals.*—significant differences in relation
to the control group, p < 0.05
Swimming—paracetamol
No significant differences for any of the exposed
animals were observed, indicating that there were
no changes in the swim ming pattern (Fig. 1b).
Swimming—propranolol
Statistical analysis of the data showed a significant
difference between animals from the control group
and those exposed to the second lowest
concentration of propranolol (0.001 mg L −1) (χ2 =
4.54, p = 0.03). At this concentration, the number of
animals that presented a typical swimming pattern
increased in relation to the control, as the number of
fish with atypical swimming pattern decreased (Fig.
2b).
Pigmentation—paracetamol
No significant differences were observed in any of
the treat mentsin relationtothe animals fromthe
control group (Fig. 1c).
Pigmentation—propranolol
Chi-square statistical analysis showed a significant
increase in number of nonpigmented organisms, for
the two highest con centrations of this drug: 0.1 mg
L−1 (χ2 = 4.26, p = 0.038) and 1 mg L−1 (χ2 = 5.6, p =
0.017) (Fig. 2c).
Histopathological and histochemical
analysis Qualitative analysis
The histological analysis of the liver of P. harpagos,
from the control group, showed that the hepatocytes
were distributed in hepatic cords of a single cellular
layer, around the sinusoidal capillaries radiating
from a central vein (Fig. 3).
Vacuoles present in the cytoplasm of hepatocytes
are pos itive for PAS reaction (Fig. 3), negative for
lipid (Sudan Black staining) and protein (Xylidine
Ponceau staining), as shown in the Supplementary
material.
Semiquantitative analysis of histopathology
Paracetamol The experimental groups presented
hepatocytes with vacuolization and red blood cell
congestion. The results of the semiquantitative
analysis of score valuesweremultiplied by the
importance factor, generating the rates of
occurrence of hepatic changes (index) for each
experimental group (Table 1).
No significant differences were observed in the
indices of this histological alteration among fish from
the control group and those exposed to different
concentrations of the drug. This set of data showed
that fish exposed to paracetamol presented a similar
frequency of occurrence of hepatic vacuolization in
all experi mental groups (df = 5,24; F = 2.827; p =
0.7266) (Fig. 4a).
Statistical analysis showed that there were no
significant differences among the animals in the
control group and those exposed to the different
concentrations of paracetamol, in terms of the
 frequency of red blood cell congestion in the blood
vessels (df = 5.24; F = 7.906; p = 0.1615) (Fig. 4b).

Fig. 3 Liver of P. harpagos. a–c

Sections stained with
hematoxylin-eosin. Note the
cord-like plaques in the liver, ar
ranged in a single layer of hepa
tocytes (dashed circle). d PAS re
action. The arrow indicates gly
cogen stores in hepatocytes.
Legend: c = cord of hepatocytes,
cv = central vein, s = sinusoid
capillary, he = hepatocyte, n =
hepatocyte nucleus, rc = red blood
cells, ct = connective tissue, b =
biliary duct

Propranolol The experimental groups presented

hepatocytes with vacuolization and red blood cell
congestion. In addition, the groups exposed to
propranolol showed alterations in tissue
cytoarchitecture and hepatocyte degeneration. The
results of the semiquantitative analysis of score
values were multiplied by the importance factor,
generating the rates of occurrence of hepatic
changes for each experimental group (Table 2).
Alteration of the tissue architecture is evidenced
by the partial or complete absence of the normal
pattern of hepato cyte array in cords around
sinusoidal capillaries (Fig. 2c, d). Although this
alteration was observed only in the groups ex posed
to the different concentrations of propranolol, while
absent in the control group, there were no
significant differ ences in their frequency of
occurrence between the experi mental groups (df =
5.24; F = 10.99; p = 0.0516) (Fig. 5a).
No significant differences were observed in the
indices of vacuoles in hepatocyte cytoplasm among
the control group and the different paracetamol
concentrations, evidencing a sim ilar frequency of
occurrence of hepatic vacuolization among all
Environ Sci Pollut Res
experimental groups (df = 5.24; F = 9.282; p =
0.0983) (Fig. 5b).
Hepatic hydropic degeneration was observed only
in the liver of the fish exposed to the two highest
concentrations of propranolol (0.1 and 1 mg L −1)
(Table 2). However, in terms of the frequency of
occurrence of this alteration, there were no
significant differences between the experimental
groups (df = 5.24; F = 3.941; p = 0.558) (Fig. 5c).
There was no difference between the animals in
 the control group and those exposed to the different Histochemical analysis
concentrations of propranolol in terms of the
frequency of occurrence of red blood cell congestion Paracetamol The semiquantitative histochemical
in the blood vessels (df = 5.24; F = analysis of the intensity of reaction for glycogen
7.7; p = 0.1736) (Fig. 5d). detected in liver hepato cytes of the P. harpagos fish
showed that there were no

Table 1 Mean and standard deviation of the occurrence rate of hepatic changes (index) in the liver of P. harpagos individuals from
the control group and exposed to different concentrations of paracetamol. N = 5 individuals per experimental group

Alteration of the liver Ctl (0 mg L−1) C1 (0.008 mg L−1) C2 (0.08 mg L−1) C3 (0.8 mg L−1) C4 (8 mg L−1) C5 (80 mg L−1)

Vacuoles in the cytoplasm of hepatocytes Discussion

Congestion of red blood cells in the vessels
1.5 ± 1 2 ± 1.63 1.5 ± 1.91 1.5 ± 1.91 1 ± 1.15 2.5 ± 1 1 ± 1.15 0
Behavioral biomarkers
± 0 0 ± 0 0.5 ± 1 1 ± 1.15 1.5 ± 1

Environ Sci Pollut Res Paracetamol exposures

Locomotor activity is one of the most important

Fig. 4 Paracetamol—frequency of the occurrence of a
vacuoles in the hepatocyte cytoplasm and b red blood cell
congestion in the fish livers of the control group and of those
exposed to paracetamol in different concentrations. Index of
vacuolated cytoplasm and red blood cell congestion in
hepatocytes from the control group and exposed to the
different paracetamol concentrations
significant differences among the control group and
the paracetamol-exposed groups at different
concentrations (df = 5.24; F = 2.649; p = 0.7539)
(Fig. 6; Table 3).
Propranolol The semiquantitative analysis of the
reaction intensity for glycogen in the liver of P.
harpagos fish showed a significant difference in the
lower concentration of propranolol (0.0001 mg L −1)
compared to the other experimental groups (df =
5.30; F = 14.66; p = 0.0119), in which there was a
decrease in the intensity of PAS positive labeling,
corresponding to glycogen stores in he patocytes
(Fig. 7; Table 4).
parameters that characterize the activity and
swimming patterns of fish, and one of the first
features that may be significantly altered by
impairments at the neuronal and neuromuscular
levels. The absence of significant alterations in
swimming patterns among all groups of fish exposed
to paracetamol suggests that this drug did not cause
evident neuronal or motor alterations in fish
subjected to the tested experimental conditions.
Similar results were obtained by Xia et al. (2017),
after evaluating the effect of paracetamol on the
locomotor activity of Danio rerio fish larvae.
However, paracetamol can indeed affect neurobe
havioral traits in sensitive organisms. Paracetamol
was shown to exert a stimulatory effect on
serotonergic descending path ways of rodents (Pini
et al. 1996), which are involved in the inhibition of
pain sensation (Jozwiak-Bebenista and Nowak
2014). Therefore, this analgesic effect of
paracetamol is in duced by the increase of
serotonin, and the experimental de pletion of
serotonin reverses the analgesic effect of paraceta
mol (Fukushima et al. 2017). This drug also has a
mild anti inflammatory effect because it is an NSAID
(nonsteroidal anti-inflammatory drug), that acts on
the inhibition of prosta glandin synthesis, by
modulating the serotonergic descending pathway
(Jozwiak-Bebenista and Nowak 2014). Such inter
ference in the metabolism and secretion of serotonin
may be relevant in behavioral terms, since this
neurotransmitter is important for the regulation of
several key functions, such as sleep, feeding, pain
perception, pleasure, and sexual stimula tion
(Winberg and Nilsson 1993; Summers and Winberg
2006). Secretion of serotonin is also involved in
maintaining homeostasis and promoting acclimation
during physiological or environmental challenges
(Chaouloff 1993). Thus, fish ex posed to
paracetamol could have their serotonergic descend
ing pathways compromised by this drug (Pini et al.
1996) in order to keep homeostasis without
presenting any significant alteration in swimming
pattern, as demonstrated in this test.
 On the other hand, the absence of neurotoxic paracetamol exposure
(Pereira et al. 2018) and behavioral effects following

Table 2 Mean and standard deviation of the occurrence rate of hepatic changes (index) in the liver of P. harpagos individuals from
the control group and exposed to different concentrations of propranolol. N = 5 individuals per experimental group

Alterations of the liver Ctl (0 mg L−1) C1 (0.0001 mg L−1) C2 (0.001 mg L−1) C3 (0.01 mg L−1) C4 (0.1 mg L−1) C5 (1 mg L−1)

Tissue architecture 0 ± 0 2 ± 0 1.2 ± 1.09 1.2 ± 1.09 0.4 ± 0.89 1.2 ± 1.09 Vacuole in the cytoplasm 1.5 ± 1 0 ± 0 0 ± 0 0.4 ±
0,89 0.8 ± 1.09 0.4 ± 0.89 Hepatic degeneration 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0.8 ± 1.78 0.8 ± 1.78 Congestion of red blood cells 1 ±
1.15 0.4 ± 0.89 0 ± 0 0 ± 0 0 ± 0 0.8 ± 1.09
Environ Sci Pollut Res

Fig. 5 Propranolol—frequency of the occurrence of a intensity of their pigmentation (Van der Salm et al.
cytoarchitecture alterations, b hepatocyte cytoplasm, c 2006; Ducrest et al. 2008; Backström et al. 2014).
hydropic degeneration, and d red blood cell congestion in the
fish livers (P. harpagos) of the control group and exposed to The here-obtained data suggest that although
propranolol in different concentrations. Index of paracetamol exposure increased the stress of fish (a
modification that was evidenced by the results
obtained from the scototaxis test, as described
is somewhat surprising. Previously published data later), this level of stress was not sufficient to mod
have shown that paracetamol may induce ify the pigmentation profile of exposed fish. The data
neurotoxicity in fish (namely, the European eel from the present study differed from the results
Anguilla anguilla) by inhibiting indirectly, via obtained by David and Pancharatna (2009), who
oxidative effects, the activity of the enzyme AChE evaluated the effects of exposure to
cytoarchitecture alterations, hepatocyte cytoplasm, hydropic
(Nunes 2011; Nunes et al. 2015). In addition, Solé et
degeneration, and red blood cell congestion in the liver from
al. (2010) demonstrated a similar effect in a study animals of the control group and exposed to different
with the marine mussel species Mytilus concentrations of propranolol
galloprovincialis. Therefore, if physiological and
behavioral changes occurred in the fish ex posed to
paracetamol, in the present study, these were not
observed in terms of swimming patterns of fish.
According to Nunes et al. (2008), dark
pigmentation in fish can be associated with different
factors that are inherently challenging for the
organism, for example, capture, environ mental
changes (including chemical stress), and as a
defense mechanism when they feel threatened.
Moreover, increased pigmentation has also a
relationship with stress responsive ness, since fish
subjected to stress (eg, social stress) increase the
 environmentally relevant concentrations of enzyme AChE (Nunes 2011; Nunes et al. 2015) by
paracetamol dur ing the embryonic development of oxidative mechanisms. This effect culminates in the
D. rerio, demonstrating that exposure to this drug change of its normal conformation, resulting in loss
resulted in anomalies in different stages of fish of its usual hydrolytic capacity, also demonstrated in
development, including changes in pigmentation the study of Solé et al. (2010) with mussels Mytilus
(depending on concentration), growth, behavior, and galloprovincialis. According to Padilla (1995),
larval survival. This suggests that, in relation to the organisms ex posed to chemical substances, which
pigmentation parameter, P. harpagos are not very inhibit the activity of AChE, showed changes in
responsive to toxic agents such as paracetamol, at behavior. The authors describe that inhibition of
least during the adult phase, or that the AChE activity may cause abnormal behavior.
environmentally relevant concentrations used in this However, the here-exposed organisms (namely
study were not sufficient to cause evident changes those exposed to the highest concentration of
in skin pigmentation. paracetamol) preferred the dark compartment.
Fish usually have a natural tendency for darker Previous data obtained by Pereira et al. (2018)
compart ments (Aranha and Caramaschi 1999; showed that exposure of individuals of P. harpagos
Wolff and Donatti 2016). This preference reflects a to levels of paracetamol that were similar to the ones
behavior of hiding from the threats posed by used in the present study did not result in
possible predators. However, an increase in cholinesterasic impairment; indeed, the authors
frequency and time in the dark compartment should reported a significant increase in ChE activity, that
reflect anxiety-promoting behavior, because the fish cannot be associated to neurotoxic effects, and from
avoids performing exploratory activity in the clear which no known behavioral changes may derive.
(white) compart ment (Maximino et al. 2010). On the
contrary, an increase in the white compartment
activity should reflect anti-anxiety be havior because
the fish can explore the tank (Maximino et al. 2010).
Anxiety may be modulated by the effects of
GABAergic and serotonergic drugs (Magno et al.
2015). The stimulatory effect that paracetamol
exerts on the serotonergic descending pathways
(Pini et al. 1996) could act on locomotor activity.
Environ Sci Pollut Res

Fig. 6 Intensity of glycogen

marking in fish liver (P.
harpagos) in the control group
and in the groups exposed to
paracetamol. Intensity of reaction
for hepatic glycogen marking that
was determined in animals of the
Propranolol
control group and exposed to exposures
different concentrations of
paracetamol. *—significant
differences relative to the control
group, p < 0.05

Kawashima et al. (2016) observed that the

serotonergic neu rons DRN (serotonergic dorsal
raphe nucleus) of D. rerio larvae act on locomotor
activity and movement-related learn ing. The
serotonergic neurons respond to the visual
movement that induces swimming. The results of
the scototaxis test of P. harpagos individuals
exposed to paracetamol are probably related to the
locomotion of the fish into the dark compartment
because of the drug-induced stress that might have
occurred at higher concentration.
One of the already described effects of In the present study, environmentally relevant
acetaminophen is the inhibition of the activity of the concentrations of propranolol increased the number
 of animals exhibiting typical swimming pattern,
showing that, at low concentra tions, the animals
were less agitated compared to the control group—
this might reflect an effect that counteracted the nat
ural anxiety caused by exposure. This possibility is
reinforced based on data already published in the
literature. Mitchell and Moon (2016) observed that,
after acute exposure of D. rerio to propranolol,
juvenile fish exposed to this drug showed re duced
anxiety, whereas fish exposed to propranolol at
embryo stage (larvae) did not exhibit anxiety
behavior in adulthood, even after being subjected to
a 6-month recovery time in water without this drug.
The β-adrenergic receptors transduce the cellular
signal of catecholamines (CAs), during its release as
part of the stress response (Gether 2000). After the
release of CAs, as a result of a stimulus, the
animal's energy reserves are mobilized by the
catecholamine binding at the β-adrenergic receptors
of the liver to support the increased energy demand
during the stress response. Therefore, exposure to a
β-blocker drug (such as propranolol) may result in a
quick mobilization of glycogen stores, ie, energy
reserves; after this short-term transient re sponse,
exposure to propranolol may decrease the body's en
ergy reserves, which would be required for the
metabolic changes involved in stress responses,
probably affecting the behavioral performance of the
animal.

Table 3 Mean and standard deviation of the frequency of PAS-positive intensity for glycogen in fish liver (P. harpagos) from the
control group and exposed to different concentrations of paracetamol. N = 4 individuals per experimental group

Liver Ctl (0 mg L−1) C1 (0.008 mg L−1) C2 (0.08 mg L−1) C3 (0.8 mg L−1) C4 (8 mg L−1) C5 (80 mg L−1) Glycogen 2.21 ± 0.5 1.56 ±

0.95 1.86 ± 0.81 1.74 ± 0.22 1.62 ± 0.47 1.86 ± 0.81

Fig. 7 Intensity of glycogen The adrenergic system exerts different functions in

marking in fish liver (P. the fish organism, such as modulation of cardiac
harpagos) in the control group
and in the groups exposed to output (Altimiras et al. 1995), metabolic regulation
propranolol. Intensity of reaction (Van Heeswijk et al. 2006), skeletal muscle yield
for hepatic glycogen marking that (Mcdonald et al. 1989), as well as the regulation of
was determined in animals of the melanophores func tioning, and consequently, color
control group and exposed to change (Burton 2002); thus, propranolol may act
different concentrations of
propranolol. *—significant directly on this specific function, as it acts
differences relative to the control pharmacologically and more broadly as a β-blocker.
group, p < 0.05 Studies have shown that the neurotransmitter
responsible for pigment aggregation in fish
melanophores is noradrenaline, which acts on
adrenoreceptors (Fujii 1961; Scheline 1963; Scott
1965;

Table 4 Mean and standard deviation of the frequency of PAS

positive intensity for glycogen in fish liver (P. harpagos) from
the con trol group and exposed to different concentrations of
propranolol (df =
The significant changes in swimming patterns Environ Sci Pollut Res
registered in animals from the group exposed to a
low propranolol concen tration (C2 = 0.001 mg L −1)
may be a hormetic response. Hormesis is
characterized by a biphasic dose-response rela
tionship, exhibiting stimulation in response to low
doses and inhibition at higher dose (Calabrese
2008). β-Blockers drugs may have a biphasic
response (hormesis) as observed in fish cell
cultures (rainbow trout RTG-2 strain), which
responded biphasically to exposure to propranolol in
an in vitro assay (NT assay, which measures the
incorporation of vital dye in lysosomes of cells) in
response to this drug (Bain and Kumar 2014).
Animals exposed to propranolol showed changes
in pig mentation at the two highest concentrations
(0.1 and 1 mg L−1), with a decrease in the number of
pigmented fish in relation to the nonpigmented fish,
so that the number of nonpigmented fish increased.
 Healey and Ross 1966; Fujii and Novales 1972).
This result has already been described for the fish
Lebistes reticulatus by Miyashita and Fujii (1975).
These authors stated that β adrenoreceptors have a
central effect on the dispersion of me lanosomes.
The intracellular mobility of melanosomes (organ
elles that produce pigment) present in melanophores
(type of chromatophore) has neuronal regulation,
which involves the dispersion of melanosomes by
the cellular cytoplasm and the aggregation of these
organelles in the periphery. The darker pigmentation
is caused by the pig ment dispersion of melano
phores, when stimulated by norepinephrine; this is a
response probably mediated by β-adrenergic
receptors which once ac tivated, lead to an increase
in cAMP, and this secondary mes senger of the
signal transduction pathway regulates the cyto
skeletal motor proteins that move the melanosomes
Liver Ctl (0 mg L−1) C1 (0.0001 mg L−1) C2 (0.001 mg L−1) C3 (0.01 mg L−1) C4 (0.1 mg L−1) C5 (1 mg L−1) Glycogen 1.88 ± 0.70
0.26 ± 0.59* 0.90 ± 0.82 1.24 ± 0.89 1.18 ± 0.60 2.33 ± 0.60
Environ Sci Pollut Res
cAMP, thus leading to the aggregation of
melanosomes. In the present study, propranolol
probably induced the aggregation of melanosomes
of melanophores at the two highest concen trations,
and the fish became less pigmented. However, this
clearer coloration in the dorsal-caudal region of the
fish ex posed to these two concentrations of
propranolol corresponds to its normal pigmentation,
which is a clear coloration under natural conditions
(Menezes et al. 2007). This fact could be related to
the anti-anxiety effect of propranolol observed in D.
rerio (Mitchell and Moon 2016), which may have
reduced the stress level of the fish to the point that
they became calm and unstressed and exhibited
their normal pigmentation.
Propranolol did not affect the stress level of the
fish, since there was no change in their preference
for the dark compart ment. None of the
concentrations tested was able to alter the behavior
of these fish, at least for this parameter. This result
was also described for D. rerio, as reported by
Magno et al. (2015) (20, 5, and 5 mg L−1) when
observing fish for 300 s. The absence of increased
stress levels of P. harpagos exposed to propranolol
could be probably related to a decrease in anx iety.
Acute exposure of D. rerio to propranolol reduced
the anxiety of juvenile fish exposed for 2 weeks to
propranolol (Mitchell and Moon 2016).
Cellular biomarkers
In the present study, the histological analysis of the
liver of P. harpagos showed an arrangement of
hepatocytes in cords around the sinusoid capillaries,
which is similar to that de scribed for the fish
Poecilia vivipara (Menezes-Faria 2009) that belongs
to the same taxonomic family as P. harpagos.
According to Akiyoshi and Inoue (2004), this cord
of the melanophores, dispersing them (Sköld et al.
2016). Lighter cutaneous pigmentation is caused by
the aggregation of mela nosomes in melanophores
(Sugimoto 2002). The signaling pathway mediated
by protein kinase C is involved in this aggregation,
leading to a decrease in the intracellular level of
cAMP, and when these secondary messengers are
in low quantity, there is an increase in the locomotor
activity of cy toskeletal dyneins, while the activity of
myosin and kinesin decrease, resulting in the
aggregation of melanosomes (Sköld et al. 2016).
However, the inhibition of β-adrenergic receptors
by pro pranolol can also lead to a decrease in the
intracellular level of
5.30; F = 14.66; p = 0.0119). N = 4 individuals per
experimental group. *—significant differences relative to the
control group, p < 0.05
arrange ment of hepatocytes is common in the more
derived teleost fish and it is similar to the
arrangements described for mam mals. This
arrangement was also described in the liver of
several species of different fish families, such as
Hoplias malabaricus (Lemes and Braccini 2004),
Hoplias lacerdae (Cruz et al. 2005), Oreochromis
niloticus (Vicentini et al. 2005), and Oligasarcus
jenynsii (Petcoff et al. 2006).
In P. harpagos, glycogen stores are mostly
present in the hepatocyte cytoplasm, as also
described in the Poecilia vivipara (Menezes-Faria
2009). According to Welsch and Storch (1973),
teleost fish contains two types of hepatocytes, one
rich in lipids, and the other rich in glycogen, with
distinct predominance among different species
(Akiyoshi et al. 2001). In P. harpagos, only
hepatocytes containing glycogen were observed.
Histopathology and histochemistry of
the liver (semiquantitative analysis)
Paracetamol The increase of vacuolization in fish
hepatocytes, which has been described in the liver
of tilapia fish
(Oreochromis niloticus) exposed to high
concentrations of paracetamol (500 mg/kg), is
considered a hepatotoxic effect of this drug (Kavitha
et al. 2011). Nevertheless, since vacuolization in P.
harpagos liver was similarly present in individuals
from all experimental groups, it is possible to hy
pothesize that paracetamol exposure was not
causative of hep atotoxic effects in the present
study.
Vacuolization in the liver is normally present in
fish popu lations at low proportions, even though
they were not exposed to chemical substances, as
observed in Poeciliia vivipara by Menezes-Faria
 (2009), or Tilapia guineensis(Guinean tilapia) and exposed to the lowest concentration of the drug
African catfish (Clarias gariepinus) (Agbohessi et al. (0.0001 mg L−1), in relation to the other experimental
2015). Thus, vacuolization is not necessarily a groups. As observed by means of the behavioral
histopatholog ical alteration induced by pollutants. biomarker analysis, in this case, a hormetic
In addition, a concentration of paracetamol that response may also have oc curred, indicating that
far exceeded the here-tested levels caused red glycogen mobilization occurred at the lower
blood cell conges tion in the liver vessels of tilapia concentration of propranolol. Probably, this mobiliza
fish (Oreochromis niloticus) (Kavitha et al. 2011), tion of glycogen was important to increase the
being considered a circulatory disorder named amount of glucose in the blood, which may be
hyperemia (Bernet et al. 1999). Nevertheless, in the involved with an increase in energy demand due to
present study, blood vessel congestion occurred in swimming activity, corroborating the results of fish
all experi mental groups in a similar way, and no behavior, in which fish exposed to the lower
exposure-related change was reported at the concentrations of propranolol had their swimming
different sublethal concentrations of paracetamol. activity increased.
This apparent blood vessel congestion in P. Marcon et al. (2015) reported that glycogen
harpagos liver probably occurred because of the depletion in hepatocytes from lambari fish (Astyanax
structure of the hepatic lobes of the fish that, given bimaculatus) in creased energy metabolism in
the absence of lobula tion in the hepatic response to exposure to the insecticide endosulfan,
parenchyma, have a slower blood flow in the vessels which altered the fish behavior during exposure
(Wolf and Wolfe 2005). (Pereira et al. 2012). Glycogen depletion was also
The absence of alteration in PAS-positive reported in the liver of the fish Tilapia guineensis
reaction for glycogen in the liver of P. harpagos (Guinean tilapia) and the African catfish Clarias
acutely exposed to paracetamol, in comparison to gariepinus(Agbohessi et al. 2015), and the authors
fish from the control group, suggests that the related this glycogen mobilization with increased
different sublethal concentrations of para cetamol energy expenditure, in response to the addi tional
were not able to induce modification in hepatic metabolic demand required for the xenobiotic
glycogen stores in the fish. On the contrary, biotrans formation process. In P. harpagos, as
individuals of the catfish Pangasius sutchi exposed swimming activity was progressively reduced as the
to a higher concentra tion of paracetamol (500 concentrations of propranolol were increased, this
mg/kg) evidenced reduction of the level of both may be one of the factors that have allowed the
glycogen and lipids in the liver (Shivashri et al. maintenance of hepatic glycogen storage at nor mal
2013). levels, like those observed in the control group.
There are two main enzyme pathways by which
Propranolol There were no significant differences glycogen can be degraded to glucose
with respect to histopathological changes among the (glycogenolysis) in the liver. One of them is
experimental groups exposed to propranolol, degradation by the enzyme glycogen phosphorylase
indicating that this drug did not in duce in the cytoplasm, which is activated by high levels of
hepatotoxicity. Despite the absence of intracel lular cAMP (Hirsimäki 1983), and the other is
hepatotoxicity at the histological level, the lower by the action of the enzyme alpha-glucosidase in the
concentration of propranolol significantly induced a lysosomal system, by means of autophagy of the
reduction in the histochemical positive reaction for glycogen deposits (Kotoulas et al. 2006). In this
hepatic glycogen. As with other vertebrates, cat context, the mobilization of glycogen in fish liver is
echolamines stimulate hepatic glycogenolysis and activated by the catecholamines that act, via G
gluconeo genesis (Fabbri et al. 1995), by means of protein signaling, in the liver energetic storage,
signal transduction pathways dependent on the leading to an increase in the release of hepatic
second messenger, cAMP (Janssens and Grigg glycogen (Danulat and Mommsen 1990; Fabbri and
1988; Janssens and Lowrey 1987). It has been Moon 2016) as a response of fish to any type of
shown that isoproterenol, another β-agonist drug, stress (Owen et al. 2007; Fabbri and Moon 2016).
increased adenylate cyclase activity in a dose- As propranolol is a β-blocker, it acts by inhibiting the
dependent man ner and was thus able to modify the cate cholamine activation of adrenoreceptors, which
intracellular concentration of cAMP (Exton 1985). in turn de creases cAMP and decreases glycogen
mobilization (Ahles and Engelhardt 2014; Fabbri and
Moon 2016). Therefore, we can observe that for the
Propranolol has been shown to specifically block highest concentration (1 mg L−1) of propranolol, P.
β adrenoreceptors of the liver of catfish (Fabbri et al. harpagos were able to maintain the glyco gen level,
1992). In which means that the sublethal concentrations used
Environ Sci Pollut Res did not affect the balance between glycogenolysis
and gluconeogenesis.
Conclusions Paracetamol exposure was only capable of inducing
responses at the behavioral level, namely by altering
contrast, a depletion of hepatic glycogen was the light/dark com partment (scototaxis). This
observed in re sponse to propranolol, but this change resulted in a clear preference of P. harpagos
change occurred only in the hepatocytes of fish for the dark compartment, but only after being
 exposed to a concentration of 80 mg L −1, which is status of wild fish inhabiting a cotton basin heavily
impacted by pesticides in Benin (West Africa). Sci Total
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of Biology in UFSCar for the infrastructural support for Chemosphere 108:334–342
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Funding information Bruno Nunes was hired under the
personal care products, a food additive and pesticides) in
program Investigador FCT, co-funded by the Human Potential
waters of Sydney estuary, Australia. Mar Pollut Bull
Operational Program (National Strategic Reference
97(1–2):56–66
Framework 2007–2013) and European Social Fund (EU).
Thanks are due to the program Pesquisador Visitante Brandão FP, Rodrigues S, Castro BB, Goncalves F, Antunes
Especial, financed by Fundação Coordenação de SC, Nunes B (2013) Short-term effects of neuroactive
Aperfeiçoamento de Pessoal de Nível Superior (CAPES), pharmaceutical drugs on a fish species: biochemical and
Brazil, with the project entitled BAvaliação dos efeitos behavioural effects. Aquat Toxicol 144:218–229
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ambiental em espécies de peixes autóctones brasileiros: uso Microsc Res Tech 58(6):481–487
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