FLUORIDE TOXICITY AND ITS EFFECTS ON GAMETOGENESIS … · Research report Fluoride 46(1)7–18...

Research reportFluoride 46(1)7–18January-March 2013

Effects of fluoride on the aquatic oligochaete Branchiura sowerbyi BeddardCasellato, Del Piero, Masiero, Covre

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FLUORIDE TOXICITY AND ITS EFFECTS ON GAMETOGENESIS IN THE AQUATIC OLIGOCHAETE BRANCHIURA

SOWERBYI BEDDARDSandra Casellato,a Stefania Del Piero,a Luciano Masiero,a Valeria Covrea

Padova, Italy

SUMMARY: The purpose of this study was to examine the tolerance of the oligochaeteBranchiura sowerbyi Beddard and its capacity to accumulate fluoride (F) by performingshort-term (96-hr LC50) and long-term experiments (18 days) at different temperatures(17ºC and 22ºC). LC50 values showed that this aquatic worm is more resistant to F thanother freshwater invertebrates, especially in the presence of sediment. Moreover, theeffects of fluoride on gametogenesis were examined by exposing this oligochaete toconcentrations 50 to 100 times above the normal of 0.1–0.3 mg F/L for five months fromthe end of February 2012 to the end of July 2012. The results revealed temporaldifferences in the gametogenesis phases of treated specimens, such as incompletespermatozoa maturation when exposed to the highest fluoride concentration. At the endof the experiment, the exposed animals were thinner and shorter than the control, andnormal cocoon deposition did not occur.

Keywords: Abnormal gametogenesis; Branchiura sowerbyi Beddard; Fluoride accumulation; Oligochaeta.

INTRODUCTION

The inorganic fluoride (F) level in freshwater varies from ≤0.1 mg/L to morethan 1 mg/L in volcanic areas,1 but nowadays local levels of F are often increasingowing to the impact of human activities, such phosphate fertilizer production,aluminum smelting, bad waste disposal, and many others industrial processes. F isfound in many common household products, including toothpastes, some vitaminsupplements, and medicines, and it is also present in nearly every food.2 Due to itsubiquity, F is ingested daily in varying amounts. However, in recent decades,serious health consequences of excessive F intake have been found, warrantingfurther precautionary measures from its consumption in high dosages.

Several studies on animal and plant phyla in aquatic environments have revealedthat safe concentrations of F are significantly different between classes, families,genera, and species, and that aquatic animals living in freshwaters are moreadversely affected by F contamination than those living in seawater because of thebioavailability of F ions (and, consequently, their toxic action) is reduced withincreasing water hardness.3 The risks to vertebrates are fairly well known, with themost significant effects on bone cells that can lead to the development of skeletalfluorosis.4 It is also now recognised that F can affect soft tissue cells, such as renal,gonadal, and neurological cells,5-7 and that its main toxic effect is due to itsinteraction with enzymes.8,9

The effect of F on animal and human reproduction has likewise become an areaof great interest, and several studies have reported toxic effects on the reproductivesystem, particularly in males.10-18 Studies on rats revealed that NaF causedalterations in testicular seminiferous tubules, such as vacuolation and denudation

aFor correspondence: Sandra Casellato, Department of Biology, University of Padova, Via U.Bassi 58/A, 35129, Italy; E-mail: [email protected]



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of spermatogenic epithelium, which disrupted spermatogenesis, reducing spermcounts and serum testosterone levels.19 Changes in the epididymal secretoryepithelium also impaired its metabolic and functional status, inhibited spermmaturation and motility, and reduced the fertility of treated animals. Negativeeffects of F on gametogenesis have been reported in many animal species as wellas in humans,20 and hence prolonged exposure to F may have serious implicationsfor fertility.

Although aquatic oligochaetes have a long history for their use in pollutionmonitoring,21-27 no data appear to have been reported on the toxicity andaccumulation of F in them. Owing to its relatively large size and prominentposterior gills,28 Branchiura sowerbyi Beddard can be easily collected andidentified, and it has a low natural mortality rate in the laboratory compared withother tubificids.29 These worms feed in the sediment, a behaviour that involves theintake of large amounts of substrate.30 Because of its size, B. sowerbyi is moreappropriate for accumulation tests than smaller oligochaeta species.31 Finally, B.sowerbyi dominates other bottom macroinvertebrates in terms of abundance andbiomass,28 and it is also a significant part of the diet of some fish and crustaceans.As far as we are aware, no previous researchers have studied effects of F ongametogenic processes in aquatic oligochaeta, so our investigation will contributeto fill these gaps.

MATERIALS AND METHODS

The specimens of B. sowerbyi used in our study came from a lily tank at theBotanic Garden of Padua University, which has been monitored for twodecades.32,33 The tank is filled by a thermal spring, and the temperature fluctuatesbetween 17°C and 27°C throughout the year. Samples of the original tank waterand the original sediment were collected, together with the oligochaetes, to set upoptimal laboratory culture and test conditions. Two hundred worms were graduallyacclimatised for one week to the test temperatures in 1-L glass containerscontinuously aerated. To evaluate the toxic effects of sodium fluoride (NaF),different experiments were performed: four short-term tests (96-hr), two long-termtests (18 days), and one test on the gametogenesis for 5 months, starting from thebeginning of February 2012.

To determine whether the temperature has an influence on F toxicity andaccumulation, the short- and long-term experiments were performed at 17±0.5ºCand 22±0.5ºC, which are the temperatures at which B. sowerbyi has the lowest andthe highest growth rate, respectively.34 The vitality of the animals was frequentlychecked using soft tweezers, and they were considered dead when there was noresponse to physical stimulation. The dead specimens were removed and kept in4% formaldehyde for F accumulation analysis. At the end of the experiments, thesurviving worms were also placed in formaldehyde and analysed.

F accumulation in the soft tissues of B. sowerbyi (as total body) was determinedby the method of Malde et al.35 For F in the sediment, the alkali fusion method ofMcQuacker and Gurney36 was used. The analyses were conducted at pH 5.5 afteradding a total ionic strength adjustment buffer (TISAB III) and using a F ion-



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selective electrode ISE F800 (0.02 mg F–/L to saturation, accuracy 0.1%, WTW).An analysis of variance (ANOVA) was carried out on F accumulation values(expressed in µg F–/g of dry weight) to determine the change in the responsevariable (Software R ver. 2.15.0). Data relationships were assessed using simplelinear regression.

Short-term experiments: In these experiments, performed with and withoutsediment, the worms were exposed to NaF at nominal concentrations of 80, 160,320, and 640 mg F–/L in glass containers covered with Plexiglas plates to preventF evaporation. The daily mortality percentage was used to estimate LC50 values atthe 95% confidence level using multifactor probit analysis (SAS softwarever.9.1.3).37

Without sediment: Fifteen worms (three replicates of five) were exposed to NaFin glass containers, filled only with the original water.

With sediment: The same number of worms were treated with NaF, introducing125 g of site sediment into each glass container.

Long-term experiments: In the long-term tests, three replicates of five worms foreach temperature were exposed to NaF at nominal concentrations of 80, 120, and160 mg F–/L, which are the concentrations at which B. sowerbyi showed thelowest mortality percentage in the short-term tests. To ensure the survival of theanimals, these long-term tests were performed only in the presence of sitesediment. Every week 10 mL of water was taken from each container andcombined with 10 mL of TISAB III for ion-selective electrode analysis. At the endof the experiment, F content analysis was performed on both the worms and thesediment.

Effects on gametogenesis: To mimic the conditions of the original sampling site,three small 10-L aquariums were prepared using the original water together withan 8-cm layer of the original sediment and the original population density (200worms/m2). Ninety specimens (30 in each aquarium), with an average length of 9–12 cm) were reared for five months and exposed to 0, 15 and 30 mg NaF/L at23±1ºC. This temperature was chosen for optimal gamete maturation.38 Thetreated and untreated specimens were checked weekly to monitor gametematuration in both male and female germinal lines. Every week some specimenswere removed and anaesthetised with 70% alcohol, after which segments from 5 to17, containing the entire sexual apparatus, were dissected using a micro-scissors.These segments were placed on a slide and gently compressed with a cover slip tolet the germinal cysts shed into the coelomatic fluid from the testes, ovaries, andseminal vesicles. In this manner, cytomorphological changes during thetransformation of spermatogonia to mature spermatozoans in males and thetransformation of oogonia to mature eggs in females could be observed under alight microscope (Leica Application Suite software with a Leica HQ DFC480camera) over the course of five months.

The nominal F concentrations were maintained by weekly renewal of the waterduring the entire treatment period. In addition, F accumulation in the sediment was



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periodically tested to quantify the real exposure level of the worms to F over time.The F concentration was determined from aliquots of water from the aquariums atthe beginning of the experiment and before every weekly renewal using the ion-selective electrode ISE F800 already described. The sediment was also monitoredby taking 0.5-g samples and analysing them by the alkali fusion method, asdescribed by McQuaker and Gurney.36 Chemical analyses were conductedperiodically (monthly) during the experiments to quantify organic matter content39

and the concentration of dissolved oxygen.40

RESULTS

Short-term experiments: B. sowerbyi demonstrated high tolerance to F,especially at 17ºC: at this temperature the mortality was lower than at 22ºC, bothwith and without sediment (Table). The mortality was also reduced in the presenceof sediment at both 17ºC and 22ºC, as evidenced by the LC50 values (Table).

Temperature influenced F accumulation, with worms treated at 22ºCaccumulating greater amounts of F in their bodies than those treated at 17ºC(Figure 1A, B). The presence of sediment enhanced the F accumulation values atboth temperatures: the animals treated with sediment accumulated double theamount of F (Figure 1A), and the accumulation trend increased with increasing Fconcentration (p<0.001).

Long-term experiments: The influence of temperature on F toxicity in B.sowerbyi was also evident also in the long-term tests. Although the same mortalitypercentage was found for all concentrations at 17ºC, it increased monotonically by1.2% for every 10 mg F–/L added (r = 0.94) at 22ºC (Figure 2).

F accumulation in B. sowerbyi showed a significant statistical relationship withthe increasing concentration of F in the water (p<0.001), and the highest valueswere observed in animals treated at 22ºC (Figure 3).

Sediment adsorbed part of the F dissolved in water, especially at 22ºC, and theaccumulation trend was directly proportional to the increasing F concentration inthe water (p<0.01) (Figure 4).

Table. LC50 values at 96 hr for B. sowerbyi at different temperatures (95% confidence limits in parenthesis)

Temperature

17±0.5°C 22±0.5°C

Sediment present

Sediment absent

Sediment present

Sediment absent

LC50 (mg F–/L) 267.63 (257.75–277.51)

91.28 (84.50–98.05)

80.07 (62.10–111.55)

61.68 (47.83–90.11)



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Figure 1. F accumulation in B. sowerbyi during short term experiments (96 hr) attemperatures of 17°C and 22°C. A) With sediment; B) Without sediment. (Error bars showthe standard deviations).

Figure 2. Mortality percentages in B. sowerbyi during long-term experiments (18 days) attemperatures of 17°C and 22°C.



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Water analyses showed that animals and sediment absorbed the most F duringthe first 6 days of the experiment, and that the highest percentage decrease of F inthe water was reached at 22ºC (Figure 5). F concentrations in the water wereclearly inversely related to increasing temperature.

Effects on gametogenesis: When the specimens of B. sowerbyi were firstexposed to NaF at the beginning of February 2012, they showed normal gonadactivity, as indicated by the increased volume of the testes and ovaries, as well asby the appearance of four to eight spermatogonial cysts in the sperm sacs of malesand large oogonia in the ovaries of females (Figure 6A). None of the specimenshad yet developed a clitellum, which appeared only after one month of NaFexposure in both the treated animals and in the controls.

Figure 4. Sediment F content at the end of the long term experiments (18 days) at temperatures of 17°C and 22°C. (Error bars show the standard deviations).

Figure 3. F accumulation in B. sowerbyi during long term experiments (18 days) at temperatures of 17°C and 22°C. (Error bars show the standard deviations).



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At the beginning of March 2012, the first 16-cell spermatogonial cysts (46 µm)(Figure 6B) appeared in the controls, which occurred one month later in the treatedanimals. On the other hand, free oocytes in the coelom were found in all thetreated and the untreated specimens at the beginning of March (Figure 6C). Theyappeared greatly enlarged (70 µm), showing more inglobing yolk globules in theircytoplasm. The first 32-cell spermatocyte cysts (60 µm) appeared at the beginningof April in the control specimens, but they did not appear until one month later inthe worms treated with either 15 mg/L or 30 mg/L of NaF (Figure 6D). The firstmature eggs were observed in the control animals. These were observed only onemonth later for those treated with 15 and 30 mg NaF/L (Figure 6E). However,development of the male germinal line occurred normally in the control animals.The 64-cell spermatocytes (Figure 6F) and the 128-cells spermatid cysts (Figure6G) were present in large numbers in the coelom cavity, and free spermatozoanswere present in some of the controls at the end of May (Figure 6H). In the treatedworms, the first spermatid cysts were not observed until one month later, and theyshowed certain deformities (Figure 6I). No free spermatozoans were found inthese specimens. At the beginning of June, some spermatozeugmata could beobserved in the seminal funnels of the control specimens, and these were alsofound one month later into the spermathecae (Figure 6J), which confirmed theyhad copulated. These structures have already been described for other tubificidspecies as sperm bundles that are transferred into the spermathecae of thecopulation partner (Figure 6K).41,42 Mature spermatozoa and spermatozeugmatawere never observed in the treated specimens, which also had fewer mature eggs,

Figure 5. Decreasing water F content during long term experiments (18 days) attemperatures of 17°C and 22°C.



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when compared with the controls. When the experiment was completed at the endof July, the treated worms appeared to be shorter and thinner than the controls.

Figure 6A–6F. A) Large oogonia in the peripherical part of the ovary; B) 16-cellsspermatogonia cyst, as indicated by the arrow; C) Large oocyte inglobing yolk globules; D)32-cells spermatocytes cyst; E) Mature egg; F) 64-cells spermatocytes cyst;



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The samples from the aquarium containing 15 mg NaF/L showed that theconcentration varied from 15 to 19 mg F–/L. In the aquarium that contained 30 mgNaF/L, the values ranged from 30 to 39.56 mg F–/L, suggesting that some of the Fwas absorbed by the sediment and was then released back into the water at eachweek of water renewal. The F concentration in the sediment increased from aninitial level of 2.59 ppm to 95.87 ppm (dry weight) in the aquarium filled with 15

Figure 6G–6K. G) 128-cells spermatid cyst; H) Free spematozoans; I) Abnormal spermatid cyst in a treated specimen; J) Spermatozeugma (sperm bundle) into the spermatheca of a specimen of B. sowerbyi after copulating, as indicated by the arrow; K) Spermatozeugma.



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mg/L of NaF and reached 122.01 ppm in the aquarium with 30 mg NaF/L,indicating that the worms were exposed to a higher concentration of F than thenominal level in the water during the experimental period. The amount ofdissolved oxygen varied between 3.9 and 5.3 mg/L, corresponding to an averagesaturation percentage of 68% at 25ºC. The mean organic matter content neverexceeded 4%, which is consistent with the level in the original sampling site.

DISCUSSION

As our data indicate, increased temperature amplified the toxicity of F: withhigher temperature, B. sowerbyi became less tolerant to this pollutant. Angelovicet al.43 demonstrated that higher temperature increases the rate of metabolism andtoxicity of F in rainbow trout, and this probably happened in our experiments,resulting in faster F absorption and faster poisoning of the enzyme systems at 22ºCthan at 17ºC. Camargo3 in his review reported similar findings of increased Ftoxicity in freshwater invertebrates, showing that the 96-hr LC50 ranges from 10.5mg F–/L for some species of crustaceans to 45 mg F–/L for the most resistantspecies of caddisfly (Chimarra marginata). B. sowerbyi showed a 96-hr LC50close to the 24-hr LC50 value of Daphnia magna, which is considered one of theless sensitive invertebrate species.44 In the absence of sediment, 50% of the wormpopulation died in water with 61.68 mg F–/L at 22ºC; the same value was reportedfor Dreissena polymorpha.45 The resistance of B. sowerbyi during short-termexposure was enhanced by the presence of the sediment. Chapman et al.,25

Casellato and Negrisolo,26 and Naqui46 used various toxicity bioassays todemonstrate that sediment reduces the bioavailability of contaminants, limiting thetoxic effects on organisms exposed to pollutants dissolved in water. This wasobserved for B. sowerbyi only in the short-term experiments. However, when theexposure time was extended to 18 days, these tubificids fed on the contaminatedsediment. Thus, their F accumulation values increased (Figure 4). Severalauthors47-49 have suggested that the contaminated sediments ingested by deposit-feeding animals should be considered the major source of the absorbedcontaminant. Therefore, sediment was an important source for the accumulation ofF in the long-term exposures because of its high adsorption capacity. We alsoobserved that absence of sediment represents a source of stress for this species,because then the worms were unable to feed.

Finally, as in studies on vertebrates showing a toxic effect of F onspermatogenesis, our results demonstrated that this toxicity of F is more commonthan commonly thought, being present in many animal groups. Prolongedexposure to F damages the gametogenic cycle in B. sowerbyi, particularly thespermatogenic one, even if the worms were exposed to concentrations that did notshow an effect from short- or medium-term exposure. Temporal differences wereobserved in the gametogenetic phases of the treated specimens when comparedwith the controls. The treated specimens did not complete spermatozoanmaturation at the highest NaF concentration used, and the number of mature eggsproduced was greatly reduced. The male line stopped maturation at the spermatidstage, which appeared abnormal and regressed. Moreover, the treated animals



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were thinner and shorter than the controls at the end of the experiment, and cocoondeposition did not occur.

Whereas F toxicity data for mammals and birds are fairly abundant, suchinformation for invertebrates has been completely lacking until now. Our researchon B. sowerbyi emphasises the importance of investigating other animal groups toexplain the exact mode of action of F and to monitor its toxic potential.

ACKNOWLEDGEMENTS

This work was supported by a grant from Padova University “Progetti di Ateneo(PRAT)–Bando 2010”.

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FLUORIDE TOXICITY AND ITS EFFECTS ON GAMETOGENESIS … · Research report Fluoride 46(1)7–18...

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