3,4,5-trihydroxybenzoic acid

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

adult fish: sub(lethal) effects

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was designed to investigate the effects of chronic waterborne exposure to the current-use phytochemicals gallic and pelargonic acids on zebrafish (Danio rerio). For this purpose, mature males and females were exposed to each type of contaminants under semi-static conditions for 1 month. Since breeding success is critical for fish species sustainability, population fecundity of the exposed adult zebrafish, with subsequent hatching time and rate were regularly evaluated. Then, a panel of biochemical, physiological, and histological biomarkers was assessed. Plasma sex steroid hormonal levels (11-ketotestosterone, 11- KT; 17 bestradiol, E2), circulating vitellogenin (Vtg) concentrations, testicular, and ovarian histology were monitored respectively in males and females at the end of experiments to investigate more thoroughly any potential impact on reproductive function. Moreover, selected liver enzyme activities were assessed as exposure biomarkers. The latter included lactate dehydrogenase (LDH) which is involved into energy production (Diamantino et al., 2001; Coelho et al., 2011), glutathione-S-transferase (GST) which plays a key role in phase II biotransformation of endogenous and xenobiotic substances, and glutathione peroxydase (GPx) that protects cells against ROS formation (van der Oost et al., 2003; Hermes-Lima, 2004).

GLP compliance:

no

Specific details on test material used for the study:

gallic acid (Acros Organics, 98% purity)

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

Stock solutions of 8 g/L gallic acid (Acros Organics, 98% purity) were prepared and renewed weekly in dechlorinated tap water.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

All fish treatment and procedures used in this study were in accordance with the general guidelines of the Council of European Communities (European Union, No 2010/63/CEE) and the French Animal Care Guidelines. Wild-type Tuebingen strain zebrafish were purchased from GIS AMAGEN (UMS 3504 CNRS/UMS 1374 INRA, Gif-sur-Yvette, France) at 3 months of age. They were housed for three additional months in the laboratory in a fish containment unit (HP Aquarium, France) filled with dechlorinated and aerated tap water (at 26618C on a 12 h:12 h light:dark cycle). They were daily fed with newly hatched Artemia nauplii and once a week with additional commercial pellets (BioMar, France). The same rearing conditions were followed during the exposure period.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

1 mo

Test temperature:

25.6 ± 0.1°C

pH:

8.2 ± 0.0

Dissolved oxygen:

6.9 ± 0.1 mg/L

Conductivity:

223.1 ± 0.5 µS/cm

Nominal and measured concentrations:

For each type of test compounds, the following nominal exposure concentrations were established: 0.05, 0.5, and 5 mg/L of substance (and control groups of 0 mg/L).

Details on test conditions:

Two weeks before experiments, groups of randomly selected 5 males and 5 females, based on morphological differences (i.e., shape of abdomen from gravid females vs males), were introduced into experimental tanks (15 L whole-glass containers
filled with 10 L of tap water, i.e., 1 fish/L). Thereafter, fish were exposed either to gallic or pelargonic acid for 1 month under semistatic conditions, i.e., 70% daily water exchange was made using peristaltic pumps connected to appropriate stock solution reservoirs of gallic/pelargonic acid (or of noncontaminated freshwater). Debris and feces were removed daily with a siphon and additional 80% water volume was exchanged two times a week to ensure steady concentrations of test compounds in each exposure tank.

Spawning, Embryonic Development, and Hatching Time
Reproductive performance of zebrafish was evaluated twice a week during the exposure period. Population fecundity was determined in this work as the average number of eggs per female per successful breeding experiment (Penglase et al., 2014).Hatching rate (%), median hatching time (dpf), and developmental abnormalities (%) were calculated for each exposure tank where a successful breeding experiment, i.e., egg production, was noticed. Mean values were then calculated over the entire exposure period according to the treatment modality (control group or exposure to gallic or nonanoic acid), and the total number of successful breeding experiments which have been reported for each treatment modality.

Reference substance (positive control):

no

Duration:

1 mo

Effect conc.:

> 0.05 - < 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: No dead fish

Duration:

1 mo

Effect conc.:

> 0.05 - < 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks on result:

other: no abnormality

Duration:

1 mo

Effect conc.:

> 0.05 - < 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: protein content

Remarks on result:

other: no significant variations

Duration:

1 mo

Effect conc.:

> 0.05 - < 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

gonadal histology

Remarks on result:

other: No significant variation in relative germ cell types for both sex and contaminant exposure conditions

Duration:

1 mo

Effect conc.:

> 0.05 - 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

fertility

Remarks on result:

other: no statistical difference between control and treatments for exposed female zebrafish

Duration:

1 mo

Effect conc.:

> 0.05 - < 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

other: lowest egg hatchability for fish exposed to 0.5 mg/L

Details on results:

Fish Condition Index
First of all, no dead fish neither any abnormality in animal behavior (swimming, feeding) could be reported during all the exposure period. Moreover, condition index was evaluated at the end of exposure as a general marker of overall fish health status (Table I). No differences were found among control and treatment groups regardless contaminant type and organisms gender (Table I).

Liver Enzyme Activities
As reported in Table I, for both male and female specimens exposed to gallic acid, no significant variations were observed in total protein content, nor in LDH and GST activities. In contrast, higher levels of total GPx activity have been measured in males and females exposed to gallic acid compared to control regardless the exposure concentration (Table I). These increases in GPx activity were significant for female zebrafish exposed to 0.05, 0.5, and 5 mg/L of gallic acid.

Gonad Development
Overall, testes and ovaries with typical organizational architecture were observed for all fish specimens from control and treatment groups. Moreover, the presence of all germ cell types could be reported in gonad sections from males and females exposed to gallic acid. Besides, a general trend has been noticed in the relative proportions of germ cells regardless contaminants’ nature and concentration. Indeed, testes were mainly composed of cell types which were at their latest stage of development (spermatids
and spermatozoids) and to a lesser extent in the spermatocyte stage. The opposite pattern was observed for ovaries with a higher relative frequency of primary growth stage oocytes (type I) with typical enlarged basophilic cytoplasm, whereas the relative
number of cortical alveolus stages (types II and III) and vitellogenic and mature follicles (grouped under “type IV” category) were lower, ranging from 10% to 15% in average. Furthermore, no significant variation was detected in relative germ cell types for both sex and contaminant exposure conditions.

Fecundity, Hatching Rate, and Embryo-Larval Development
As reported in Table II, there was no statistical difference in mean fecundity between control and treatments for gallic acid exposed female zebrafish. The evaluation of overall hatching rates indicated that the lowest egg hatchability was observed for fish exposed to 0.5 mg/L of gallic acid with 32.068.2% of total egg survival (Table II). In addition, no significant differences were found between control and treatments regarding embryonic development malformation rate (Table II).

TABLE I. Fish condition index, liver total protein content, and enzyme activities of male (M) and female (F) zebrafish after 1 month exposure to gallic acid, and the control group (0 mg/L of test substance)

		Control	Gallic acid
		0 mg/L	0.05 mg/L	0.5 mg/L	5 mg/L
Fish condition index	M	0.80±0.03	0.73±0.03	0.79±0.02	0.80±0.03
	F	0.82±0.02	0.88±0.02	0.83±0.03	0.90±0.06
Liver total protein content (mg/g fresh weight)	M	77.5±16.8	88.7±13.6	96.3±6.4	84.0±14.3
	F	86.2±8.5	62.8±13.6	77.2±11.6	49.2±7.8
LDH activity (U/mg protein)	M	1839±416	1322±135	1922±182	2116±143
	F	512±100	661±57	446±40	516±75
GST activity (U/mg protein)	M	359±50	225±16	286±29	332±31
	F	168±11	223±27	169±19	175±16
GPX activity (U/mg protein)	M	446±111	507±58	593±97	668±101
	F	39±12	214±87*	171±56*	214±61*

Data are expressed as mean±SE. Asterisks indicate significant differences against control (p<0.05).

 

TABLE II. Evaluation of reproductive parameters in zebrafish over 1 month exposure to gallic acid in the concentration range of 0, 0.05, 0.5, and 5 mg/L of test substance

	Control	Gallic acid
	0 mg/L	0.05 mg/L	0.5 mg/L	5 mg/L
Fecundity (mean egg number/ female/spawning event)	12.1±3.3	8.0±1.5	16.5±4.5	24.4±4.9
Median hatching time (dpf)	3±0.3	4.1±0.3	4.5±0.3	4.4±0.3
Hatching rate (%)	49.7±18	65.9±17	32.0±8.2	59.4±10
Developmental abnormalities (%)	13.7±8.0	3.6±0.8	10.0±3.5	5.3±1.7

Data have been averaged over the exposure period (mean±SE).

hpf: hours post fecundation; dpf: days post fecundation.

 

 

 

Validity criteria fulfilled:

not specified

Conclusions:

This work aimed at investigating the sublethal effects of a long-term waterborne exposure of zebrafish to gallic acid. Mature fish were exposed to gallic acid at the concentrations of 0, 0.05, 0.5 and 5 mg/L during
one month under semi-static conditions. Fecundity, hatching rate and median hatching time were regularly evaluated. Circulating sex hormone levels (11 ketotestosterone 211 KT, 17 bestradiol -E2-), plasma
vitellogenin (Vtg), and gonad histology were monitored in males and females after exposure. Lactate dehydrogenase (LDH), total glutathione peroxydase (GPx) and glutathione-S transferase (GST) activities
were assessed as enzymatic biomarkers of exposure in fish liver. Significant increases of GPx activity were reported in females exposed to gallic acid regardless the contamination level. Moreover,
5 mg/L gallic acid induced a decrease in 11-KT levels for males. However, no histological alteration in gonads neither significant variation in reproductive performances were detected following zebrafish exposure to gallic acid. Additional investigations concerning the mode of application and the environmental fate of this substance may warrant their further use in freshwater systems at concentrations compatible with biocidal/allelochemical effects.

Executive summary:

This work aimed at investigating the sublethal effects of a long-term waterborne exposure of zebrafish to gallic acid. Mature fish were exposed to gallic acid at the concentrations of 0, 0.05, 0.5 and 5 mg/L during

one month under semi-static conditions. Fecundity, hatching rate and median hatching time were regularly evaluated. Circulating sex hormone levels (11 ketotestosterone 211 KT, 17 bestradiol -E2-), plasma

vitellogenin (Vtg), and gonad histology were monitored in males and females after exposure. Lactate dehydrogenase (LDH), total glutathione peroxydase (GPx) and glutathione-S transferase (GST) activities

were assessed as enzymatic biomarkers of exposure in fish liver. Significant increases of GPx activity were reported in females exposed to gallic acid regardless the contamination level. Moreover,

5 mg/L gallic acid induced a decrease in 11-KT levels for males. However, no histological alteration in gonads neither significant variation in reproductive performances were detected following zebrafish exposure to gallic acid. Additional investigations concerning the mode of application and the environmental fate of this substance may warrant their further use in freshwater systems at concentrations compatible with biocidal/allelochemical effects.

Description of key information

The existing study has not been designed to determine a NOEL but investigates the sublethal effects of a long-term waterborne exposure of zebrafish to gallic acid. Mature fish were exposed to gallic acid at the concentrations of 0, 0.05, 0.5 and 5 mg/L during one month under semi-static conditions. Significant increases of Glutathione Peroxydase (GPx) activity were reported in females exposed to gallic acid regardless the contamination level. Moreover, 5 mg/L gallic acid induced a decrease in 11-KT levels for males. However, no histological alteration in gonads neither significant variation in reproductive performances were detected following zebrafish exposure to gallic acid. Additional investigations concerning the mode of application and the environmental fate of this substance may warrant their further use in freshwater systems at concentrations compatible with biocidal/allelochemical effects.

Based on these endpoints, gallic acid is not classified for aquatic hazards.