The Embryotoxicity of Some Phenol Derivatives on Zebrafish, Danio rerio

Document Type: Research Paper

Authors

1 Atatürk University Engineering Faculty, Department of Environment Engineering, Erzurum, Turkey

2 Atatürk University Science Faculty, Department of Biology, Erzurum, Turkey

Abstract

The existence of toxicants in ecosystems has been increased dramatically in recent years, especially in aquatic environments. Phenols and chlorinated phenol derivatives are toxic industrial compounds. Phenols and derivatives are known to be environmental contaminants. In the present study, 2,4-Dichlorophenol, 2-Chlorophenol and substituted phenol were tested for embryotoxicity and mortality in a four-day period using zebrafish, Danio rerio embryos. Tested phenol derivatives caused teratogenicity and embryo mortality in the embryos. The semi static 48-h LC50 (median lethal concentration) value for Substituted-phenol was 13.850 mg L-1; the corresponding values for 2-Chlorophenol and 2,4-Dichlorophenol were 8.378 mg L-1 and 6.558 mg L-1, respectively. The endpoints are incomplete eyes, head and tail, heart and chorda deformity, yolk sac edema, tail curvature, shrunken eyes, lordosis, delayed hatching, weak pigmentation, heart edema and non-pigmentation after exposure to the compounds. 2,4-Dichlorophenol was found to be more toxic than the others. This paper is the first to describe the relative toxicity of a suite of phenols in the early life stages of zebrafish.

Keywords


The Embryotoxicity of Some Phenol Derivatives on Zebrafish, Danio rerio

 

Zeynep Ceylan1, Turgay Sisman2*, Hatice Dane2, Şeymanur Adil2                                  

 

1. Atatürk University Engineering Faculty, Department of Environment Engineering, Erzurum, Turkey

2. Atatürk University Science Faculty, Department of Biology, Erzurum, Turkey

 

* Corresponding author’s E-mail: tsisman@atauni.edu.tr

ABSTRACT

The existence of toxicants in ecosystems has been increased dramatically in recent years, especially in aquatic environments. Phenols and chlorinated phenol derivatives are toxic industrial compounds. Phenols and derivatives are known to be environmental contaminants. In the present study, 2,4-Dichlorophenol, 2-Chlorophenol and substituted phenol were tested for embryotoxicity and mortality in a four-day period using zebrafish, Danio rerio embryos. Tested phenol derivatives caused teratogenicity and embryo mortality in the embryos. The semi static 48-h LC50 (median lethal concentration) value for Substituted-phenol was 13.850 mg L-1; the corresponding values for 2-Chlorophenol and 2,4-Dichlorophenol were 8.378 mg L-1 and 6.558 mg L-1, respectively. The endpoints are incomplete eyes, head and tail, heart and chorda deformity, yolk sac edema, tail curvature, shrunken eyes, lordosis, delayed hatching, weak pigmentation, heart edema and non-pigmentation after exposure to the compounds. 2,4-Dichlorophenol was found to be more toxic than the others. This paper is the first to describe the relative toxicity of a suite of phenols in the early life stages of zebrafish.

 

Key words: Substituted phenol, 2-Chlorophenol, 2,4-Dichlorophenol, zebrafish, embryotoxicity.

INTRODUCTION

An increasing diversity of environmental pollutants are entering the aquatic ecosystem and causing potential long-term negative effects on organisms in aquatic system (Livingstone 1998; Livingstone 2001).  Phenols and chlorinated derivatives are industrial toxic compounds (Pera-Titus et al. 2004). These pollutants originate from different sources such as the effluents of the petrochemical industries, and various chemical manufacturing industries (herbicides, pesticides, solvents, paints, plastics, etc.) (Arana et al. 2001).  Phenols are widely used in the synthetic chemical industry, dying, mining and agriculture, and they are organic pollutants. Phenols and their chlorinated derivatives are resistant to biodegradation processes. Therefore, phenols may accumulate in aquatic biota and negatively affect all aquatic organisms (Czaplicka 2004). Chlorophenols (CPs) are extensively used as by-product of bleaching in paper mills, as wood treatment agent and for biocide production. CPs also show a very wide distribution in the environment (Stringer & Johnston 2001). Di-, tri- and penta-chlorophenol are classified as priority pollutants by USEPA since CPs have adverse effects on human and wildlife, such as chronic toxicity, mutagenicity and carcinogenicity (Ramamoorthy & Ramamoorthy 1997). 2-Chlorophenol (2CP) is on the priority pollutant list of the USEPA and is used in pulp, paper and pesticide industries (Dec et al. 2003).  The USEPA has also recommended restricting 2CP and 2,4-Dichlorophenol (2,4-DCP) concentrations in freshwaters to below 4380 and 2020-ug L-1, respectively (USEPA 1980a; USEPA 1980b). It is considered that industrial waste discharge including chlorophenols creates major water pollution (Krijgsheld & Van der Gen 1986). Some phenols involved in surface waters are 2-CP, 2,4-DCP and 2,4,6-TCP (Scow et al. 1982). The most abundant phenol in aquatic environments is 2,4-DCP (House et al. 1997). 2,4-DCP was determined in drinking water supplies in the USA and the highest detected concentration was 36-ug L-1 (Shackelford & Keith 1976). The toxicity of phenol and derivatives were widely studied on some invertebrates and vertebrates. For example, Prati et al. (2000) and Qiao et al. (2006) reported that phenols induced genotoxic effects in animals and human. Although previous studies were focused on the general toxicity of phenols, few studies investigated the developmental toxicity of the compounds. Therefore, the aim of this work was to evaluate the developmental toxicity of phenol derivatives by means of zebrafish bioassay.

Zebrafish, Danio rerio (Hamilton, 1822) is a valuable test organism, especially for toxicity research. The fish has transparent embryos and short spawning time (Nagel 2002). In addition, zebrafish is a model organism, and has many advantages such as being easy to maintain and breed, inexpensive and providing rapid assay for acute and chronic toxicity (Westerfield 2007). The objectives of the research were i) to determine the median lethal concentrations (LC50) of substituted phenol, 2-Chlorophenol and 2,4-Dichlorophenol for 48 hpf embryos of zebrafish, ii) to study the teratogenic effects of the phenol derivatives on the development of the fish, and iii) to compare the toxicity of the three phenol compounds.

 

MATERIALS AND METHODS

Chemicals

Substituted phenol (SP), 2-Chlorophenol (2-CP) and 2,4-Dichlorophenol (2,4-DCP), were obtained from Sigma-Aldrich (Darmstadt-Germany). Before starting the test, stock solutions of the phenol derivatives were prepared by dissolution in low-conductivity water prepared from a MiliQ water treatment system, and stock solutions were stored at 5 °C.

 

Fish culture and egg production

Adult zebrafish samples were provided by Atatürk University Fisheries Faculty Research Centres for Aquarium Fish. Before the experiments, the fish were acclimatised for 14 days. Twenty healthy adult fish were placed in each aquarium with a photoperiod 14:10 light and dark cycle. Dechlorinated municipal water was used in aquaria and maintained at 27±1 ºC. The water was renewed at 1/3 ratio every week.

The fish were fed with dry flake food twice a day, and live feed was fed once every two days. Breeding groups were formed and placed separately in small spawning aquarium which were equipped with glass balls on the bottom. No air filters were used in the spawning aquaria and fish were not fed during breeding. When the light was turned on, spawning was induced in the morning. Half an hour later, healthy and fertilized eggs were collected and placed in fresh embryo medium (Hank’s solution), and incubated at 27±1 ºC until treatment.

 

 

Embryotoxicity and Teratogenicity

The fish embryos were exposed to phenol concentrations to determine the 50% lethal concentrations (LC50). The experimental concentrations of SP, 2CP and 2,4-DCP (2, 4, 8, 16 and 32 mg L-1) were inspired by previous work (Nagel 2002). Hank’s solution was used as control medium. At approximately 3 hour post-fertilization (hpf), blastula stage embryos were selected under a stereomicroscope in Hank’s solution. The exposure was begun after placing the fertilised eggs into the test solutions. Twenty embryos were used in each group including control. Each experiment was repeated three times (total 360 embryos for one phenol compound). Phenol exposure to embryos was performed in a glass petri dish (10 cm diameter) containing 50 ml test solution at 27±1 ºC with photoperiod of 14:10-h light/dark cycle in a precision incubator until 96 hpf.

Test solutions were renewed with fresh solutions every 24-h (semi-static test condition). Every 12-h, the embryos were observed and scored for lethal and sublethal effects using a microscope with a digital camera. Embryo-larva stages were categorized as described by Kimmel et al. (1995). According to Lammer et al. (2009), lethal and sublethal effects were determined. Lethal abnormalities (coagulation, missing heartbeat, somites, tail detachment and spontaneous movement) were determined for each group. Then, dead embryos or larvae were immediately removed and recorded at each observation time. Some teratogenic malformations (incomplete eyes, head and tail, heart and chorda deformity, yolk sac edema, tail curvature, lordosis, heart edema, non-pigmentation) were also recorded. The embryos and larvae were scored for malformations at 72-hpf according to the rating scale by Padilla et al. (2011).

Each embryo or larva was marked for various categories such as curved spine, non-hatching and edema on the scale. Each of the categories included a number of abnormalities scored as yes/no or as degree. Total Malformation Index (MI) was calculated using the scores from each category for each embryo. MI values of 0-3 MI indicated as normal; 4-6 values as slightly abnormal, and values above 7 as obviously deformed embryo or larva.

Statistical analysis

SPSS (version 20.0) software programme was used for statistical analysis. The 48-h LC50 values were determined by probit analysis. Statistical differences in the rate of embryo lethality and number of abnormal embryos/larvae were evaluated with One-Way ANOVA. All data were expressed as mean ± standard deviation (SD). For multiple comparisons, analysis of variance was used followed by Dunnett’s test. p

 

 

RESULTS

Calculated 48-h LC50 values by probit 95% confidence interval (CI) for SP, 2CP and 2,4-DCP were 13.850, 8.378 mg L-1 and 6.558 mg L-1 for zebrafish embryos, respectively. According to the values, 2,4-DCP was more toxic than 2CP and SP. LC50 values of the phenolic compounds for zebrafish embryos were firstly reported in this study (Figs. 1, 2 and 3).

 

Fig. 1. Dose-response curves used to calculate the LC value. LC curve of SP (95% confidence limits).

 

The developmental rate was slow in experimental groups comparing to control group. Table 1 shows that some developmental parameters such as gastrulation and somite completion, optic cup formations, spontaneous contraction, tail detachment, heartbeat, blood circulation, pigmentation of and hatching in zebrafish embryos occurred at the slowest rate in living embryos exposed to sublethal concentrations of SP, 2CP and 2,4-DCP. It was found that phenol derivatives reduced the number of hatched embryos. Also, the larvae were found to stay within the chorion and never hatched with high phenol concentrations at the end of exposure. Zebrafish embryos showed over 50% mortality in 48-h at high concentrations of the phenols. Mortality was not found to be significantly different from each other with low concentrations of the phenols. The frequency of dead embryos was distributed throughout the 48-h exposure period. Lethal and teratogenic effects were recorded at 96-hpf (Tables 2, 3 and 4). Especially, it was observed that the percentage of affected and dead embryos increased with high phenol concentrations. Table 2 shows that 16 and 32 mg L-1 SP caused embryo mortality within two days, reaching 80% mortality at 16 mg L-1.

 

Fig. 2. Dose-response curves used to calculate the LC value. LC curve of 2CP (95% confidence limits).

 

Fig. 3. Dose-response curves used to calculate the LC value. LC curve of 2,4-DCP (95% confidence limits).

 

 

 

Table 1. The Rates of development in zebrafish embryos.

Developmental perioda

Age

Cleavage

1.5 hpf

75 % epiboly

8 hpf

Pharyngula

24 hpf

Hatching

48 hpf

Early larva

80 hpf

Control

1.6 ± 0.20

8.6 ± 1.13

25.1 ± 2.21

49.0 ± 3.06

81.0 ± 3.29

SP (8 mg L-1)

1.7 ± 0.15

12.0 ± 0.42*

32.6 ± 3.45*

57.5 ± 4.80*

108.5 ± 5.89*

2CP (4 mg L-1)

1.7 ± 0.10

16.1 ± 0.76*

37.5 ± 4.86*

66.4 ± 5.13*

110.3 ± 8.56*

2,4DCP (4 mg L-1)

1.6 ± 0.10

18.9 ± 0.91*

46.6 ± 4.60*

79.2 ± 7.34*

131.1 ± 9.58*

a Described by  Kimmel et al. (1995).

The values are presented as mean ±SD. “hpf” shows hour of development periods. *Significantly different from control at p < 0.05

 

 

 

Table 2. Adverse effects of SP in the embryos at 96-hpf.

 

Control

2 mg L-1

4 mg L-1

8 mg L-1

16 mg L-1

32 mg L-1

Number of teratogenic embryos

3

3

6

20

5

0

Number of  dead embryos

3

6

9

26

43

60

Number of affected embryos

6

9

15

46

48

60

Number of normal embryos

54

51

45

14

12

0

% Teratogenic embryos

5.0 ± 1.2

5.0 ± 1.5

10 ± 1.0

33.3 ± 2.9

8.3 ± 1.6

0

% Dead embryos

5.0 ± 1.1

10 ± 1.7

15 ± 2.6

43.3 ± 4.6

71.7 ± 6.5

100 ± 0

% Affected embryos

10 ± 1.0

15 ± 2.5

25 ± 2.7

76.7 ± 6.7*

80 ± 6.1*

100 ± 0*

% Normal embryos

90 ± 5.8

85 ± 5.2

75 ± 6.6

20 ± 2.4

20 ± 4.1

0

* Significantly different from control at p < 0.05.

 

At two highest concentrations of 2CP, many of the early life stage abnormalities were persistent and resulted in embryo mortality in the next days, reaching 90% (Table 3). All embryos showed teratogenicity in 2CP group coagulated at the end of 96 hpf. A dose-response relationship was detected in the percentage of embryos with lethal and teratogenic effects after exposure to 2,4-DCP  (Table 4). At the 4 and 8 mg L-1, teratogenic effects were observed at 96-hpf, whereas at 16 and 32 mg L-1 these effects were found at 48-hpf. During the early life stages of development (< 48-h), observed abnormalities included incomplete eyes, head and tail development (Fig. 4B), heart and chorda deformity (Fig. 4D).

After hatching, clear macroscopic aberrations included yolk sac edema, tail curvature and shrunken eyes (Fig. 5B), lordosis (Fig. 5C), delayed hatching and weak pigmentation (Fig.5D), heart edema, tail curvature and depigmentation (Fig. 5E) observed in all treated embryos. No incidence of malformations was observed in the controls (Fig. 4A and 4C, Fig. 5A). The most dominant abnormalities were detected as chorda malformations. All of teratogenic embryos and larvae died within 1-2 days after 96-hpf.

 

 

Table 3. Adverse effects of 2-CP in the embryos at 96-hpf.

 

Control

2 mg L-1

4 mg L-1

8 mg L-1

16 mg L-1

32 mg L-1

Number of teratogenic embryos

2

5

16

19

8

0

Number of  dead embryos

2

4

7

24

47

60

Number of affected embryos

4

9

23

43

55

60

Number of normal embryos

56

51

37

17

5

0

% Teratogenic embryos

3.3 ± 0.2

8.2 ± 1.5

26.6 ± 2.0

31.6 ± 1.9

13.3 ± 1.6

0

% Dead embryos

3.4 ± 0.3

6.6 ± 0.3

11.6 ± 1.6

40.6 ± 3.6

78.3 ± 4.9

100

% Affected embryos

6.7 ± 0.5

15.2 ± 3.5

38.3 ± 2.2*

71.6 ± 4.0*

91.6 ± 5.3*

100*

% Normal embryos

93.3 ± 6.2

85.3 ± 5.2

61.6 ± 4.6

28.3 ± 2.1

8.3 ± 1.4

0

* Significantly different from control at p < 0.05.

 

Table 5 shows that mean total Malformation Index (MI) for 2 mg L-1 phenol congeners were not statistically different from the control groups. However, the mean total MI increased in 72-hpf embryos at 4, 8 and 16 mg L-1 phenol concentrations. The embryos in control and 2 mg L-1 phenols were normal because they were scored between 0 and 3 for MI. The other embryos were scored between 4 and 6, and were slightly abnormal (in 4 and 8 mg L-1 phenols); and in the 16 mg L-1 phenols, embryos scored above 7 were absolutely deformed. MI could not be calculated for embryos in 32 mg L-1 because all of them died in 72-hpf.

 

 

 

Fig 4. Photomicrograph ofmalformations in zebrafish embryos exposed to 16 mg L-1 SP, 8 mg L-1 2CP and 4 mg L-1 2,4-DCP. A) 24-hpf  normal embryo showing somites, eyes, head and chorda B) 24-hpf abnormal embryo showing incomplete eyes, head and tail development C) 48-hpf normal embryo showing pigmentation and normal chorda. D) 48-hpf abnormal embryo showing heart and chorda deformations (48-hpf). C: chorda, Ch: chorion, E: eyes, H: head, He: heart, T: tail, YS: yolk sac.

 

Table 4. Adverse effects of 2,4-DCP in the embryos at 96-hpf.

 

Control

2 mg L-1

4 mg L-1

8 mg L-1

16 mg L-1

32 mg L-1

Number of teratogenic embryos

2

5

18

11

5

0

Number of  dead embryos

1

5

11

34

50

60

Number of affected embryos

3

10

29

45

55

60

Number of normal embryos

57

50

31

15

5

0

% Teratogenic embryos

3.3 ± 0.5

8.3 ± 1.5

30.0 ± 2.0

18.5 ± 2.9

8.3 ± 2.9

0

% Dead embryos

1.6 ± 0.1

8.2 ± 2.5

18.3 ± 2.6

56.6 ± 5.6

83.3 ± 7.2

100

% Affected embryos

5.3 ± 0.6

16.6 ± 1.8*

48.3 ± 4*

75.6 ± 5*

91.6 ± 7.9*

100*

% Normal embryos

95.6 ± 6.9

83.3 ± 4.0

51.6 ± 5.6

25.3 ± 2.6

0.83 ± 0.3

0

* Significantly different from control at p < 0.05.

 

Table 5. Malformation Index values of three phenol congeners in zebrafish embryos at 72-hpf.

 

 

2 mg L-1

4 mg L-1

8 mg L-1

16 mg L-1

32 mg L-1

 

SP

1.8 ± 0.4

3.6 ± 0.5*

5.0 ± 1.0*

7.5 ± 1.0*

0

 

2CP

2.0 ± 0.5

4.0 ± 0.6*

5.6 ± 0.6*

8.6 ± 1.6*

0

 

2,4-DCP

2.5 ± 0.9

4.6 ± 0.5*

5.6 ± 1.0*

8.6 ± 1.0*

0

Control

1.7 ± 0.2

 

 

 

 

 

* Significantly different from control at p < 0.05.

 

 

Fig 5. Photomicrograph ofmalformations in zebrafish larvae exposed to 16 mg L-1 SP, 8 mg L-1 2CP and 4 mg L-1 2,4-DCP. A) 72 hpf normal hatched larva with normal body structure, pigmentation and well-developed eyes. B) 72-hpf abnormal larva showing tail curvature, yolk sac edema and shrunken eyes. C) 72-hpf larva showed lordosis. D) Abnormal larva in chorion showed delayed hatching and weak pigmentation (72-hpf). E) Abnormal larva showing heart edema, tail curvature and non-pigmentation (72-hpf). C: chorda, Ch: chorion, E: eyes, H: head, He: heart, S/O: sacculus/otoliths, T: tail, YS: yolk sac.

 

 

DISCUSSION

Phenol and phenolic substances are considered as the main pollutant because of the toxic effects. The substances can also be accumulated in living organisms. Their contamination in aquatic system could pose potential threat to aquatic organisms. Therefore, the studies on the ecotoxicology of the chemicals are of important. In the current study, developmental toxicities of SP, 2-CP and 2,4-DCP were investigated by zebrafish bioassay. It was found that SP, 2-CP and 2,4-DCP had important effects on the early life stage of zebrafish embryos. One of the important effects was death. Other sub-lethal effects were incomplete eyes, head and tail, heart and chorda deformity, yolk sac edema, tail curvature, shrunken eyes, lordosis, delayed hatching, weak pigmentation, heart edema and depigmentation. The most toxic chemical to 48-hpf zebrafish embryo was 2,4-DCP, followed in decreasing order by 2CP and SP. Similar order of toxicity for the compounds on fish was shown in the literature. The toxic effect of SP can be understood by looking at its LC50 values which are less than others. For example, 96-h phenol LC50 value for 28-to 43-d-old Oryzias latipes was calculated as 38.3 mg L-1 (Holcombe et al. 1995).  Also, the 48-h LC50 value of phenol for Japanese medeka (Oryzias latipes) was 24.1 mg L-1 (Rice et al. 1997). Fogels & Sprague (1977) reported that phenol (flow-through) 48-h LC50s were 11.6 mg L-1 for rainbow trout, 30.9 mg L-1 for zebrafish and 36.3 mg L-1 for flagfish. Phenol (flow-through) 96-h LC50 value was 29 mg L-1 for 30-d-old fathead minnow (Phipps et al. 1981). In a static assay for 96-h, LC50 was determined as 40 mg L-1 for male Poecilia reticulata (Colgan et al. 1982).  Moraes et al. (2015) found that the phenol LC50 values for 96-h were 15.08 and 32.56 mg L-1 for Ictalurus punctatus (channel catfish) and Piaractus mesopotamicus (pacu) respectively. Our semi-static 48-h LC50 value of SP (substituted phenol) was 13.850 mg L-1 for 48-hpf zebrafish embryos and the value was consistent with the above literatures about fish.

In Figs. 2 and 3, the 48-h LC50 values obtained for 2CP and 2,4-DCP are lower than 10 mg L-1. The results are consistent with the results of previous studies. In a previous study, it was shown that LC50 and EC50 values for 120-hpf zebrafish were 1.11, 2.45 mg L-1 and 0.74, 1.53 mg L-1 for 2,4,6-trichlorophenol and 2,4-DCP, respectively (Zhang et al. 2018). The 48-h LC50 values of 2CP and 2,4-DCP for  fathead minnow (Pimephales promelas) were determined as 8.3 and 6.7 mg L-1 respectively (Blum & Speece 1991). The 24-h LC50 value of 2CP in goldfish was 16 mg   L-1, while 24-h LC50 value of 2,4-DCP was 7.8 mg L-1 in the same fish (Kobayashi et al. 1979). Small differences in LC50 values obtained from bioassays of acute toxicity tests may be due to various factors including fish life stage, fish body size, physicochemical properties of water, the absorption rate and detoxification mechanisms among species (Bucher & Hofer 1993; Rand et al. 1995; Saha et al. 1999). Fish are sensitive to various phenol concentrations ranging from 5.02 mg L-1 to 178 mg L-1. The 96-hour LC50 value was 5.02 mg L-1 for rainbow trout and 2.5-h LC50 was 85 mg L-1 for goldfish (McLeay, 1976;Kishino & Kobayashi 1995). The 96-h LC50 values were determined in marine fish from 5.6 mg L-1 to 30.6 mg L-1 (Kondaiah & Murty 1994).  The toxic effects of phenol on aquatic organisms were extensively studied. For example, 5- to 9-day LC50s were 0.04 to 11.2 mg L-1 phenol for amphibians (Birge et al. 1980). Bernadini et al. (1996) reported that LC50 value was calculated as 178 mg L-1 phenol for Xenopus embryos.

Our results showed that the phenol compounds caused mortality and delayed hatching along with other malformations in zebrafish embryos. In a study related to zebrafish and chlorophenols, it was reported that the average number of spawned eggs of adult zebrafish exposed to 0.3 mg L-1 2,4-DCP significantly decreased and it reduced hatching success of the fish eggs (Ma et al. 2012). Previously, Sawle et al. (2010) reported disruption of neurogenesis in zebrafish embryos with 2,4-DCP toxicity. In the same study, it was also reported that the 72-h LC50 and EC50 values of 2,4-DCP for zebrafish embryos were 38.9 and 10.8 µM, respectively. Phenols are also known to have lethal and teratogenic effects on other aquatic organism embryos. It was shown that phenols significantly reduced growth of larval fathead minnows at 0.25 mg L-1 and spawning at 0.62 mg L-1 concentrations (Dauble et al. 1983). In a previous study, it was observed that Xenopus embryos exposed to 5 mg L-1 of phenol grew more slowly and died in 3 weeks (Dumpert 1987). Paisio et al. (2009) showed that phenol produced teratogenic effects such as axial flexure, persistent yolk plug, irregular forms, acephalism, edema, axial shortening and different abnormalities in Bufo arenarum embryos (stage 25) at 150 mg L-1, while lethal effects occurred at 183 mg L-1 after 96-h treatment. Bernadini et al. (1996) also reported that phenol caused serious malformations (generalized edema, intestinal and ocular malformations) in Xenopus embryos. These studies are in agreement with our teratogenic results.

Other toxicological effects of phenol compounds in several fish species were also reported. The toxic effects were haematological alterations in Dicentrarchus labrax (Roche & Boge 2000) and Ictalurus punctatus (Moraes et al. 2015), genotoxicity in Scophthalmus maximus (Bolognesi et al. 2006), carcinogenesis and mutagenesis in zebrafish (Yin et al. 2006), endocrine disruption in common carp (Kumar & Mukherjee 1988), and metabolism imbalance in Brycon amazonicus (Hori et al. 2006).

Phenols have adverse effects on aquatic life. Limited information is available regarding the mechanism of toxicity of phenols and their derivatives. It is known that phenols contribute to the loss of activity of some biochemical reactive enzymes. 2-CP induced reactive oxygen species (ROS) generation in fish (Luo et al. 2006). 2,4-DCP caused a range of oxidative damage both to proteins and lipids (Han et al. (1998). Also, phenol increases the formation of free radicals. The radicals reduce antioxidant capacity, leading to significant oxidative damage of important molecules such as DNA, protein and lipids (Murray et al. 2007). The pathway of phenol induced ROS generation was proposed as a mitochondrion NADH electron chain - dependent process and the negative pathway could lead to mitochondrion damage (Luo et al. 2008). The damage is most destructive to living organisms. It is considered that teratogenic fish embryos may be affected by phenols in the same way. However, other approaches are needed to explain developmental toxicity of phenols.

The pollution of phenol and their derivatives are common problems faced by worldwide population and ecosystems.

 

Exceeding the standard levels in environment was not new issue for developed or developing countries (Gami et al. 2014). The studies of environmental levels of these and other phenols have been carried out for the last 30 years (Jin et al. 2012). The USEPA (1989) reported that the phenol level in environmental waters should not exceed 3.5 mg L-1 to protect human and animal health. At times phenols were determined at higher concentrations than these limits. For example, phenol concentrations ranging from 0.4 to 2.28 mg L-1 were found in some river waters (Paisio et al. 2009). This study showed both mortality and teratogenic effects at ecologically-relevant SP concentrations (8 and 16 mg L-1). USEPA (1980b) reported chlorophenol concentrations ranging between 68 and 125 mg L-1 with the 2,4-DCP content ranging as high as 89% of the total in manufacturing effluent areas. Also, House et al. (1997) reported that concentrations ranging from -1 of 2,4-DCP in surface waters were determined in several countries. According to the measured concentrations of 2,4-DCP in the ecosystem and our selected concentrations of 2CP and 2,4-DCP, aquatic organisms should not be affected by the compounds now. The experimental concentrations of 2CP and 2,4-DCP used in the current study caused significant toxicity in zebrafish embryos after 48-h exposure. This study showed that phenols caused developmental toxicity in zebrafish, and the other toxic effects should be considered in future pollution research.

Arana, J, Tello-Rendon, E, Dona-Rodriguez, JM, Valdes do Campo, C, Herrera-Melidan, JA, Gonzalez-Diaz, O & Perez-Pena, J 2001, Highly concentrated phenolic wastewater treatment by heterogeneous and homogeneous photocatalysis: mechanism study by FTIR-ATR. Water Science and Technology, 44: 229–236.

Bernadini, G, Spinelli, O, Presutti, C, Vismara, C, Bolzacchini, E, Orlando, M, Settimi, R 1996, Evaluation of the developmental toxicity of pesticide MCPA and its contaminants phenol and chlorocresol. Environmental Toxicology and Chemistry, 15: 754–760.

Birge, WJ, Black, JA & Kuehne, R 1980, Effects of Organic Compounds on Amphibian Reproduction. 121. Water Resources Institute, University of Kentucky, Lexington, KY, USA.

Blum, DJW & Speece, RE 1991, A database of chemical toxicity to environmental bacteria and its use in interspecies comparisons and correlations. Research Journal of the Water Pollution Control Federation,63: 198-207.

Bolognesi, C, Perrone, E, Roggieri, P, Pampanın, DM, Sciutto, A 2006, Assessment of micronuclei induction in peripheral erythrocytes of fish to xenobiotics under controlled conditions. Aquatic Toxicology, 78: 93-98.

Bucher, F & Hofer, R 1993, Histopathological effects of sublethal exposure to phenol on two variously pre-stressed populations of bullhead (Cottus gobioL.). Environmental Contamination and Toxicology, 51: 309-316.

Colgan, PW, Cross, JA, Johansen, PH 1982, Guppy behavior during exposure to a sublethal concentration of phenol. Bulletin Environmental Contamination and Toxicology, 28: 20–27.

Czaplicka, M 2004, Sources and transformations of chlorophenols in the natural environment. Science of the Total Environment, 332: 21–39.

Dauble, DD, Barraclough, SA, Bean, RM & Fallon, WE 1983, Chronic effect of coal-liquid dispersions on fathead minnows and rainbow trout. Transactions of the American Fisheries Society, 112: 712–719.

Dec, J, Haider, K, Bollag, JM 2003, Release of substituents from phenolic compounds during oxidative coupling reactions. Chemosphere, 52: 549–556.

Dumpert, K 1987, Embryotoxic effects of environmental chemicals: test with the South African Clawed Toad (Xenopus laevis). Ecotoxicology and Environmental Safety, 13: 324–338.

Fogels, A & Sprague, JF1977, Comparative short-term tolerance of zebrafish, flagfish, and rainbow trout to five poisons including potential reference toxicants. Water Research,11: 811–817.

Gami, AA 2014, Phenol and its toxicity. Journal of Environmental Microbiology and Toxicology, 2: 11-24.

Han, SK, Ichikawa, K & Utsumi, H 1998, Quantitative analysis for the enhancement of hydroxyl radical generation by phenols during ozonation of water. Water Research, 32: 3261–3266.

Holcombe, GW, Benoi,t DA, Hammermeister, DE, Leonard, EN & Johnson, RD1995, Acute and long-term effects of nine chemicals on the Japanese medaka (Oryzias latipes). Archives Environmental Contamination and Toxicology, 28: 287–297.

Hori, TSF, Avilez, IM, Inoue, LK & Moraes, G 2006, Metabolical changes induced by chronic phenol exposure in matrinxã Brycon amazonicus (teleostei: characidae) juveniles. Comparative Biochemistry and Physiology C, 143: 67-72.

House, WA, Leach, D, Long, JL, Cranwell, P, Smith, C, Bharwaj, L, Meharg, A, Ryland, G, Orr, DO & Wright, J 1997, Micro-organic compounds in the Humber Rivers. Science of the Total Environment, 194–195: 357–371.

Jin, X, Gao, J, Zha, J, Xu, Y, Wang, Z, Giesy, JP & Richardson, KL 2012, A tiered ecological risk assessment of three chlorophenols in Chinese surface waters. Environmental Science and Pollution Research, 19: 1544–1554.

Kimmel, CB, Ballard, WW, Kimmel, SR, Ullmann, B & Schilling, TF 1995, Stages of embryonic development of the zebrafish. Developmental Dynamics, 203: 253–310.

Kishino, T & Kobayashi, K 1995, Relation between toxicity and accumulation of chlorophenols at various pH, and their absorption mechanism in fish. Water Research, 29: 431-42.

Kobayashi, K, Akitake, H & Manabe, K 1979, Relation between toxicity and accumulation of various chlorophenols in Goldfish. Bulletin of the Japanese Society for the Science of Fish, 45: 172-175.

Kondaiah, K & Murty, AS 1994, Avoidance behaviour test as an alternative to acute toxicity test. Bulletin Environmental Contamination and Toxicology, 53: 836-43.

Krijgsheld, KR & Van der Gen, A 1986, Assessment of the impact of the emission of certain organochlorine compounds on the aquatic environment. Chemosphere, 15: 825-860.

Kumar, V & Mukherjee, D 1988, Phenol and sulfide induced changes in the ovary and liver of sexually maturing common carp, Cyprinus carpio. Aquatic Toxicology, 13: 53-59.

Lammer, E, Carr, GJ, Wendler, K, Rawlings, JM, Belanger, SE & Braunbeck, T 2009, Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comparative Biochemistry and Physiology-Part C: Toxicology and Pharmacology, 149: 196–209.

Livingstone, DR 1998, The fate of organic xenobiotics in aquatic ecosystems: quantitative and qualitative differences in biotransformation by invertebrates and fish. Comparative Biochemistry and Physiology A, 120: 43–49.

Livingstone, DR 2001, Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Marine Pollution Bulletin, 42: 656–666.

Luo, YSu, Y, Wang, XR & Tian, Y 2006, 2-chlorophenol induced ROS generation in freshwater fish Carassius auratus based on the EPR method. Chemosphere, 65: 1064–1073.

Luo, Y, Su, Y, Wang, XR & Tian, Y 2008, 2-chlorophenol induced hydroxyl radical production in mitochondria in Carassius auratus and oxidative stress – An electron paramagnetic resonance study. Chemosphere, 71: 1260-1268.

Ma, Y, Han, J, Guo, Y, Lam, PKS, Wu, RSS, Giesy, JP, Zhang, X & Zhou, B 2012, Disruption of endocrine function in in vitro H295R cell-based and in in vivo assay in zebrafish by 2,4-dichlorophenol. Aquatic Toxicology, 106-107: 173-181.

McLeay, DJ 1976, A rapid method for measuring the acute toxicity of pulp mill effluents and other toxicants to salmonid fish at ambient room temperature. Journal of the Fisheries Research Board of Canada, 233: 1303-1331.

Moraes, FD, Figueiredo, JSL, Rossi, PA, Venturini, FP & Moraes, G 2015, Acute toxicity and sublethal effects of phenol on hematological parameters of channel catfish Ictalurus punctatus and pacu Piaractus mesopotamicus. Ecotoxicology and Environmental Containation, 10: 31-36.

Murray, AR, Kisin, E, Castranova, V, Kommineni, C, Gunther, MR & Shvedova, AA 2007, Phenol-induced in vivo oxidative stress in skin: Evidence for enhanced free radical generation, thiol oxidation, and antioxidant depletion. Chemical Research in Toxicology, 20: 1769-1777.

Nagel, R 2002, DarT: The embryo test with the zebrafish Danio rerio-a general model in ecotoxicology and toxicology. Altex, 19: 38–48.

Padilla, S, Hunter, DL, Padnos, B, Frady, S & MavPhail, RC 2011, Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables. Neurotoxicology and Teratology, 33: 624–630.

Paisio, CE, Agostini, E, Gonzales, PS & Bertuzzi, ML 2009, Lethal and teratogenic effects of phenol on Bufo arenarum embryos. Journal of Hazardous Materials, 167: 64-68.

Prati, M, Biganzoli, E, Boracchi, M, Tesauro, M, Monetti, C & Bernardini, G 2000, Ecotoxicological soil evaluation by FETAX. Chemosphere, 41: 1621–1628.

Pera-Titus, M, Garcya-Molina, V, Banos, MA, Gimenez, J & Esplugas, S 2004, Degradation of chlorophenols by means of advanced oxidation processes: a general review. Applied Catalysis B: Environmental, 47: 219–256.

Phipps, GL, Holcombe, GW & Fiandt, JT1981, Acute toxicity of phenol and substituted phenols to the fathead minnow. Bulletin Environmental Contamination and Toxicology, 26: 585–593.

Qiao, M, Wang, C, Huang, S, Wang, D & Wang, Z 2006, Composition, sources, and potential toxicological significance of PAHs in the surface sediments of the Meiliang Bay, Taihu Lake, China. Environment International, 32: 28–33.

Ramamoorthy, S & Ramamoorthy, S 1997, Chlorinated organic compounds in the environment: regulatory and monitoring assessment. CRC press.

Rand, GM, Wells, PG & Mccarty, LS 1995, Introduction to aquatic toxicology. In: Rand, G.M. (ed), Fundamentals of aquatic toxicology, 2nd edition. Boca Raton, FL: CRC Press.

Rice, PJ, Drewes, CD, Klunertanz, TM, Bradrury, SP & Coats, JR 1997, Acute toxicity and behavioral effects of Chlorpyrifos, Permethrin, Phenol, Strychnine, and 2,4-Dinitrophenol to 30-day-old Japanese Medaka (Oryzias latipes). Environmental Toxicology and Chemistry, 16: 696–704.

Roche, H & Bogé, G 2000, In vivo effects of phenolic compounds on blood parameters of a marine fish (Dicentrarchuslabrax). Comparative Biochemistry and Physiology-Part C: Toxicology and Pharmacology, 125: 345-353.

Saha, NC, Bhunia, F & Kavıraj, A 1999, Toxicity of phenol to fish and aquatic ecosystems. Bulletin Environmental Contamination and Toxicology, 63: 195-202.

Sawle, AD, Wit, E, Whale, G & Cossins, AR 2010, An information-rich alternative, chemicals testing strategy using a high definition toxicogenomics and zebrafish (Danio rerio) embryos. Toxicological Sciences, 118: 128-139.

Scow, K, Goyer, M & Perwak, J 1982, Exposure and risk assessment for chlorinated phenols (2-chlorophenol, 2, 4-dichlorophenol, 2, 4, 6-trichlorophenol). Cambridge, MA: Arthur D. Little. EPA, 440: 4-85.

Shackelford, WM & Keith, LH, 1976, Frequency of organic compounds identified in water (Vol. 1). Environmental Protection Agency, Office of Research and Development, Environmental Research Laboratory, Analytical Chemistry Branch.

Stringer, R & Johnston, P 2001, Chlorine and the environment: an overview of the chlorine industry. Springer Science & Business Media.

USEPA, 1980a, Ambient Water Quality Criteria for 2-chlorophenol.Report No. EPA No: 440/5-80-034. U.S. Environmental Protection Agency, Office of Water Regulations and Standards, Criteria and Standards Division, Washington, DC.

USEPA, 1980b, Ambient Water Quality Criteria for 2,4-Dichlorophenol. Report No. EPA/ 440/5-80-042. U.S. Environmental Protection Agency, Office of Water Regulations and Standards, Criteria and Standards Division, Washington, DC.

USEPA 1989, Generalized methodology for Conducting Industrial reduction evaluations TREs, EPA 600/2-88/070.

Westerfield, M 2007, The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio). 5th Edition, University of Oregon Press, Eugene.

Yin, D, Gu, Y, Li, Y, Wang, X & Zhao, Q 2006, Pentachlorophenol treatment in vivo elevates point mutation rate in zebrafish p53 gene. Mutation Research, 609: 92-101.

Zhang, Y, Liu, M, Liu, J, Wang, X, Wang, C, Ai, W, Chen, S & Wang, H 2018, Combined toxicity of triclosan, 2, 4-dichlorophenol and 2, 4, 6-trichlorophenol to zebrafish (Danio rerio). Environmental Toxicology and Pharmacology, 57: 9-18.