**3. Invertebrate model animals**

#### **3.1. Study on** *Drosophila melanogaster*

*Drosophila melanogaster* remains as one of the popular organism in studying the effects of BPA on eukaryotic biological system. The study on *Drosophila* includes change in gene expression profile, change in behaviour and nervous system, alteration in juvenile growth and develop‐ ment, history traits and fecundity and metabolism.

#### *3.1.1. Effects on life history traits and developmental event*

In comparison to other studies on effects of BPA on biological aspects in *Drosophila melanogaster*, adequate references are available on the researches on *Drosophila* life history traits. The effects of BPA on growth and development in *Drosophila* were observed, which demon‐ strated a statistically significant increase in larval growth for the low‐dose treatment group (0.1 mg/L), but not in the high‐dose treatment group (10 mg/L). BPA exposure caused an increase in body size in treated flies at 48, 72 and 96 hours following egg laying (AEL), sug‐ gesting a non‐monotonic dose response. The increase in growth rate found for all treatment groups was associated with a statistically significant increase in food intake observed at 72‐hour AEL. Furthermore, it was observed that the increased growth rate was coupled with an earlier onset of pupariation and metamorphosis, resulting from increased activity of insu‐ lin/insulin growth factor signalling (IIS) in *Drosophila*. Thus, this suggests that BPA exerts its effects through disruption of endocrine signalling in *Drosophila* since the timing of the onset of pupariation in *Drosophila* is controlled through the complex interaction of the IIS and the ecdysone signalling pathways. All these observations suggest that the effect is probably due to disruption of insulin‐like signalling in cellular system [95].

Another study on life history traits of *Drosophila* [96] obtained some contradiction to the above‐mentioned observation. The author reported a delay in both the mean pupation and the mean maturation times in treated group. In that experiment, larvae of *D. melanogaster* were exposed to three different concentrations: 0.1, 1 and 10 mg/L BPA. In the 0.1 and 1 mg/L exposed groups, the mean offspring numbers were significantly less than that of the control groups, indicating that mean fecundity was significantly decreased. Thus, administration of BPA in both food and through body wall absorption resulted in altered fecundity [96]. Mean decrease in fecundity as compared to control in *Drosophila* exposed to BPA is also evident in the work of Atli et al. [96]. William et al. [97] have reported that BPA exposure causes inhibition of lipolysis during starvation, leading to significantly increased lipid content after 24 hours of fasting. Furthermore, it also suppresses the expression of insulin‐like peptide in *Drosophila,* indicating that BPA may inhibit lipid recruitment during starvation in *Drosophila*.

#### *3.1.2. Effects on behaviour and nervous system*

*2.2.3. Other miscellaneous effects*

12 Bisphenol A Exposure and Health Risks

was the target organ of BPA.

**3. Invertebrate model animals**

**3.1. Study on** *Drosophila melanogaster*

ment, history traits and fecundity and metabolism.

*3.1.1. Effects on life history traits and developmental event*

to disruption of insulin‐like signalling in cellular system [95].

BPA can alter sex ratio of zebrafish by inducing feminization of the fry [92]. Zebrafish embryos exposed to BPA also showed signs of feminized brains [86]. Kinch et al. [93] investigated mor‐ phological changes to developing zebrafish caused by exposure to BPA including changes in body length, pericardia (heart) and the head. Na et al. [94] observed a significant damage in the liver of zebrafish after 96 hours of exposure to BPA. This result further confirmed that liver

*Drosophila melanogaster* remains as one of the popular organism in studying the effects of BPA on eukaryotic biological system. The study on *Drosophila* includes change in gene expression profile, change in behaviour and nervous system, alteration in juvenile growth and develop‐

In comparison to other studies on effects of BPA on biological aspects in *Drosophila melanogaster*, adequate references are available on the researches on *Drosophila* life history traits. The effects of BPA on growth and development in *Drosophila* were observed, which demon‐ strated a statistically significant increase in larval growth for the low‐dose treatment group (0.1 mg/L), but not in the high‐dose treatment group (10 mg/L). BPA exposure caused an increase in body size in treated flies at 48, 72 and 96 hours following egg laying (AEL), sug‐ gesting a non‐monotonic dose response. The increase in growth rate found for all treatment groups was associated with a statistically significant increase in food intake observed at 72‐hour AEL. Furthermore, it was observed that the increased growth rate was coupled with an earlier onset of pupariation and metamorphosis, resulting from increased activity of insu‐ lin/insulin growth factor signalling (IIS) in *Drosophila*. Thus, this suggests that BPA exerts its effects through disruption of endocrine signalling in *Drosophila* since the timing of the onset of pupariation in *Drosophila* is controlled through the complex interaction of the IIS and the ecdysone signalling pathways. All these observations suggest that the effect is probably due

Another study on life history traits of *Drosophila* [96] obtained some contradiction to the above‐mentioned observation. The author reported a delay in both the mean pupation and the mean maturation times in treated group. In that experiment, larvae of *D. melanogaster* were exposed to three different concentrations: 0.1, 1 and 10 mg/L BPA. In the 0.1 and 1 mg/L exposed groups, the mean offspring numbers were significantly less than that of the control groups, indicating that mean fecundity was significantly decreased. Thus, administration of BPA in both food and through body wall absorption resulted in altered fecundity [96]. Mean decrease in fecundity as compared to control in *Drosophila* exposed to BPA is also evident BPA causes [98] behavioural modifications in *Drosophila melanogaster,* which, in turn, sug‐ gests intuitively the role of environmental risk factors for the behavioural impairments like autism and attention deficit hyperactivity disorder (ADHD) in human. The study revealed disturbance in the locomotion patterns of BPA‐exposed *Drosophila* that may relate to the decision‐making and the motivational state of the animal. Furthermore, an increase in repeti‐ tive behaviour and disturbance in grooming behaviour and abnormal social interaction of *Drosophila* following BPA exposure were seen.

A recent study conducted by Streifel [99] shows that administration of BPA in the prenatal environment had significant impacts on some aspects of *Drosophila* behaviour, which includes increased time spent in seeking behaviour, increased numbers of peristaltic contractions, increased linear as well as angular movement, decrease in turn angle value as well as poten‐ tially significant impacts on motor nerve morphology. These findings suggest implication of BPA as ubiquitous neurotoxin that acts upon the delicate process of neurodevelopment.

#### *3.1.3. Effects on global gene expression profile*

Alteration in gene expression profile in *Drosophila* has been studied by Branco et al. [100]. The authors reported that the effects due to BPA on genome‐wide gene expression of *D. melanogaster* can be enhanced by the ingestion of high dietary sugar. The authors have found that acute and chronic exposure to BPA causes gross downfall in transcription of testis‐specific genes and overexpression of ribosome‐associated genes across tissues. In addition, it causes alteration of transposable elements that are specific to the ribosomal DNA loci, suggesting that nucleolar stress might implicate in BPA toxicity. This observation suggests that BPA and dietary sugar might functionally interact, with consequences to regulatory programmes in both reproductive and somatic tissues [100].

#### **3.2. Study on other invertebrate model**

As compared to vertebrates, the number of research works regarding BPA exposure on inver‐ tebrates is minimum. Invertebrates are frequently used as bioindicators for endocrine‐disrupt‐ ing chemicals. Research suggests that some invertebrates appear to be quite sensitive to BPA, and effects have been documented even at environmentally relevant concentrations [101].

#### *3.2.1. Effects on life history traits and developmental events*

A study conducted by Lemos et al. [102] revealed that low BPA concentrations disrupt the endocrine function of terrestrial arthropod *Porcellio scaber* by causing a sex‐ratio shift. In this study, endocrine system‐related chronic effects were identified at a lower dose of BPA than the concentration having acute toxic effects on isopods, indicating impairment of molting, incomplete ecdysis.

The effects of various concentrations of BPA on the development of two sea urchin species *Hemicentrotus pulcherrimus* and *Strongylocentrotus nudus* were examined [103]. This study suggested that the sensitivity of sea urchin embryos and juveniles to endocrine disrupter chemicals changes during the stages of development. The development in the first 12 hours following fertilization up to the morphogenesis of embryo was found to be most sensitive. Even higher concentrations of BPA exposure (>300 mg/L) resulted in developmental arrest and mortality in the sea urchin *Paracentrotus lividus* [104].

Studies on lepidopteran corn stalk borer *Sesamia nonagrioides* revealed that BPA induces vari‐ ous developmental disorders through interfering effect in ecdysteroidal pathway [105] and over expression of heat‐shock proteins [106]. Study on freshwater insect *Chironomus riparius* showed that adult emergence times were significantly delayed on moderate BPA exposure [107]. Marcial et al. (2003) and Watts et al. [108, 109] found that the marine copepod *Tigriopus japonicus* showed developmental inhibition at a very low concentration of BPA (0.1 mg/L). However, it is unclear if these effects have any long‐term impacts in adult life. Experimental exposure to higher concentration of (11.4 mg/L) BPA for 1 hour caused premature larval meta‐ morphosis in the marine polychaete worm *Capitella capitata* [110].

A study conducted on *Hydra vulgaris* by Pascoe et al. [111] pointed that the structure and physiology of polyps were adversely affected at concentrations greater than 42 μg/L BPA. Also, inhibition of regeneration ability was recorded above 460 μg/L BPA concentration. The results indicate that signalling processes necessary for the control and regulation of cell move‐ ment and differentiation during normal development, regeneration and sexual reproduction in *H. vulgaris* are not disrupted by BPA at low environmentally relevant concentrations.

#### *3.2.2. Effects on reproductive system and fecundity*

As far as published literatures are concerned, several studies have been conducted to unravel the adverse effects of BPA on reproductive systems and reproductive functioning in various invertebrate animals. In the study of Manshilha et al. [112], an increased fecundity (neonates per female), in comparison with the negative control group (100.3 ± 1.6%), was observed when daphnids were cultured and allowed to breed in the polycarbonate (PC) containers (145.1 ± 4.3%–264.7 ± 3.8%) for single and multiple generations. A strong dose‐dependent ecotoxico‐ logical effect was evident, and it was suggested that BPA leached from plastic materials acts as functional estrogen *in vivo* at very low concentrations. In contrast, neonate production by daphnids cultured in polypropylene and non‐PC bottles was slightly but not significantly enhanced (92.5 ± 2.0 to 118.8 ± 1.8%). Multigenerational tests also demonstrated magnification of the adverse effects, not only on fecundity but also on mortality of the species. Reproductive impairment in Daphnia due to exposure to BPA is also evident in the study by Tišler et al. [113].

Andersen et al. [6] found an increase in egg production in copepod *Acartiatonsa* exposed to 20 μg BPA/L. Moreover, inhibition in normal development at BPA concentrations above environmentally relevant levels (100 mg/L) was also evident. At extremely high exposures (16,000–80,000 mg/L), abnormal growth and inhibition of gemule germination was found in freshwater sponges *Heteromyenia sp.* and *Eunapius fragilis* [114]. A study conducted by Oehlmann et al. [115] on freshwater snail *Marisa cornuarietis* and of the marine prosobranch *Nucella lapillus* revealed that BPA affects the reproductive system and has a negative impact on snails even at nominal concentration, that is, 1 μg/L. Affected *Marisa* females were desig‐ nated as 'superfemales' and were characterized by the presence of additional female organs, hyperplasia of the accessory pallial sex glands, malformations of the pallial oviduct causing increased female mortality and a strong stimulation of oocyte and spawning mass production. In these follow‐up studies, Oehlmann et al. [116] tried to bridge several gaps in knowledge by conducting additional experiments. Here, the authors confirm the previous results and addi‐ tionally conclude that the occurrence of superfemales is associated with adverse effects on reproduction and survival, even at sub‐micrograms per litre concentrations of BPA (NOEC, 7.9 ng/L; EC10, 13.9 ng/L). However, if snails are exposed to BPA under conditions that maxi‐ mize the reproductive output, particularly during the spawning season or at elevated temper‐ atures, the induction of superfemales is at least partially masked. The superfemale induction is probably mediated by binding of BPA with estrogen receptor, because the response can completely be reversed by coexposure to potent estrogen inhibitors. Furthermore, the extreme BPA sensitivity of *M. cornuarietis* and other prosobranch snails probably due to higher affinity of the compound for the estrogen receptor in this species was compared. Overall, the results suggest that BPA imposes a potential hazard for prosobranch population in the field even at environmentally relevant concentrations. Experimentally determined EC50 values of BPA for different invertebrate model organisms have been given in **Table 5**.

#### *3.2.3. Effects on gene expression profile*

study, endocrine system‐related chronic effects were identified at a lower dose of BPA than the concentration having acute toxic effects on isopods, indicating impairment of molting,

The effects of various concentrations of BPA on the development of two sea urchin species *Hemicentrotus pulcherrimus* and *Strongylocentrotus nudus* were examined [103]. This study suggested that the sensitivity of sea urchin embryos and juveniles to endocrine disrupter chemicals changes during the stages of development. The development in the first 12 hours following fertilization up to the morphogenesis of embryo was found to be most sensitive. Even higher concentrations of BPA exposure (>300 mg/L) resulted in developmental arrest

Studies on lepidopteran corn stalk borer *Sesamia nonagrioides* revealed that BPA induces vari‐ ous developmental disorders through interfering effect in ecdysteroidal pathway [105] and over expression of heat‐shock proteins [106]. Study on freshwater insect *Chironomus riparius* showed that adult emergence times were significantly delayed on moderate BPA exposure [107]. Marcial et al. (2003) and Watts et al. [108, 109] found that the marine copepod *Tigriopus japonicus* showed developmental inhibition at a very low concentration of BPA (0.1 mg/L). However, it is unclear if these effects have any long‐term impacts in adult life. Experimental exposure to higher concentration of (11.4 mg/L) BPA for 1 hour caused premature larval meta‐

A study conducted on *Hydra vulgaris* by Pascoe et al. [111] pointed that the structure and physiology of polyps were adversely affected at concentrations greater than 42 μg/L BPA. Also, inhibition of regeneration ability was recorded above 460 μg/L BPA concentration. The results indicate that signalling processes necessary for the control and regulation of cell move‐ ment and differentiation during normal development, regeneration and sexual reproduction in *H. vulgaris* are not disrupted by BPA at low environmentally relevant concentrations.

As far as published literatures are concerned, several studies have been conducted to unravel the adverse effects of BPA on reproductive systems and reproductive functioning in various invertebrate animals. In the study of Manshilha et al. [112], an increased fecundity (neonates per female), in comparison with the negative control group (100.3 ± 1.6%), was observed when daphnids were cultured and allowed to breed in the polycarbonate (PC) containers (145.1 ± 4.3%–264.7 ± 3.8%) for single and multiple generations. A strong dose‐dependent ecotoxico‐ logical effect was evident, and it was suggested that BPA leached from plastic materials acts as functional estrogen *in vivo* at very low concentrations. In contrast, neonate production by daphnids cultured in polypropylene and non‐PC bottles was slightly but not significantly enhanced (92.5 ± 2.0 to 118.8 ± 1.8%). Multigenerational tests also demonstrated magnification of the adverse effects, not only on fecundity but also on mortality of the species. Reproductive impairment in Daphnia due to exposure to BPA is also evident in the study by Tišler et al. [113]. Andersen et al. [6] found an increase in egg production in copepod *Acartiatonsa* exposed to 20 μg BPA/L. Moreover, inhibition in normal development at BPA concentrations above

and mortality in the sea urchin *Paracentrotus lividus* [104].

morphosis in the marine polychaete worm *Capitella capitata* [110].

*3.2.2. Effects on reproductive system and fecundity*

incomplete ecdysis.

14 Bisphenol A Exposure and Health Risks

Change in expression pattern of genes and alteration in RNA expression pattern due to BPA exposure are also within the scientific interest. Planelló et al. [117] studied the effects of BPA on the expression of some selected genes, including housekeeping, stress‐induced and hor‐ mone‐related genes in *C. riparius* larvae. They found that exposure to BPA at a concentration of 3 mg/L for 12–24‐hour exposure did not influence the levels of ribosomal RNA or those


**Table 5.** Summarized presentation showing experimentally determined effective concentration (EC50) and no effect concentration (NOEC) of BPA on different invertebrate animals [103].

of mRNAs for both L11 or L13 ribosomal proteins which were selected as representative of housekeeping genes involved in ribosome biogenesis. Nonetheless, BPA treatment induced the transcription of the HSP70 gene. Interestingly, BPA causes significant increase in transcript of the ecdysone receptor (EcR), suggesting that BPA can selectively affect the expression of the ecdysone receptor gene suggesting a direct interaction with the insect endocrine system.

Significant level of DNA strand break has been detected in snail *Potamopyrgus antipodarum* under exposure to BPA [118]. DNA‐damaging effect of BPA on aquatic insect *C. riparius* has also been reported by Martinez‐Paz et al. [119].
