In this study, we tested the effects of three of the main BPA analogs (BPAF, BPF, BPS) and their mixture on the diatom
P. tricornutum. The growth curves were mainly affected by BPAF during the last part of the exposure. In addition, also the BPS caused a reduction of the cell density at day 9. The BPF did not cause any effect, while in the MIX treatment a general reduction of growth was observed, even if non-significantly. Other studies indicate that many algal species are very tolerant to BPA exposure in term of cell growth. Indeed, the EC
50 values are in the order of mg/L [
37]. However, some species can be more sensitive, such as the marine dinoflagellate
Alexandrium pacificum, which showed the growth drastically inhibited by BPA at a concentration of 2 µg/L [
38]. Our results are in accordance with those of Ding et al. [
14] that reported no significant effects on the growth rate of
Chlorella vulgaris after exposure to environmentally relevant concentrations (≤0.5 mg/L) of BPS and BPA, even if the growth decreased at higher concentrations. In other studies, the tested concentrations showed effects on the growth curve; however tested concentrations were higher than the environmental levels. Indeed, Tišler et al. [
13] recorded a value of 50% inhibition of growth in
Desmodesmus subspicatus after 72 h of exposure to both BPA and BPF (22.1 and 19.6 mg/L, respectively). In that study, BPAF showed a 50% of inhibition of growth at 3.0 mg/L, that is 1000 times higher than the concentration tested in the present study. Similarly, the growth of microalga
Tetraselmis sp. was inhibited at 2.34, 4.69, 9.38 and 18.75 mg/ L of BPA after 24, 48, 72 and 96 hours of exposure [
39]. In a recent study on
Tetrahymena thermophila, several bisphenols were tested, namely BPA, BPAF, bisphenol B (BPB), bisphenol E (BPE) and BPS. The authors reported that there was a reduction of growth caused by BPA (13.0 μM), BPAF (13.0 μM), BPB (2.6 and 13.0 μM) and BPE (2.6 and 13.0 μM) after 30 hours of exposure, and these responses were maintained after 60 hours except for BPE treatments [
40]. We observed that both cell diameter and cell volume were altered by the factors growth phase and its interaction with treatment. In detail, both diameter and volume increased during the time and reached the highest values after seven days of exposure, then they decreased. Recently, Li et al. [
40] observed that bisphenols caused a decrease in cell size of
Tetrahymena thermophila. Indeed, BPA (2.6 and 13.0 μM), BPAF (13.0 μM), BPB (13.0 μM) and BPS (13.0 μM) caused a significant reduction in cell volume after 12 hours of exposure in the adaptive growth phase and the trend was maintained after 30 hours of exposure. Furthermore, after 60 hours of exposure in the stable growth phase the cell volume was reduced in all the treatments, except for the BPE at a concentration of 2.6 μM [
40].
P. tricornutum was used in another study to evaluate the effects of six different bisphenols BPA, BPS, BPAP, BPAF, BPFL and BPC tested various concentrations, namely 5, 20, 40, 80, 150 and 300 μM [
41]. The authors observed that the growth of this microalga was inhibited by all the concentrations at all times of exposure, except for bisphenol C at 5 μM and 72 h. The authors ranked the toxicity of bisphenols for this species as follows: BPC < BPA < BPS < BPFL ≈ BPAF ≈ BPAP after 72 hours of exposure. In the same study, the authors evaluated the effects of bisphenols also on other two species of microalgae:
Tetraselmis suecica, that was the most sensitive species, and
Nannochloropsis gaditana, that was the species most resistant to bisphenols even if some effects have been observed also on this species [
41]. Interestingly the authors observed that almost all the bisphenols tested caused a reduction of cell volume of
P.
tricornutum, apart from BPS, which provoked a significant increase for this parameter at the highest concentrations, and BPFL that did not cause any effect. On the contrary,
T. suecica cells showed a significant increase in cell size, mainly at higher concentrations and after 72 h of incubation, while
N.
gaditana was less affected than the other two species. An alteration of the growth curve was also observed in the alga
Chlorella pyrenoidosa exposed to BPA that caused an increased cell density at 2 mg/L on the first day of exposure, while the other treatment groups (5, 8, 11 and 15 mg/L) showed an inhibitory effect. However, on the 4th day, also the exposure to 2 mg/L caused growth inhibition [
11]. In that study, BPS caused an increased density of algal cells at 5 mg/L, while higher concentrations (10, 15, 20 and 40 g/L) had an inhibitory effect. In addition, the combined effect of BPA and BPS in the mixture treatment was like that of the single compounds [
11]. Concentrations of BPA lower than the previous ones, were tested in
Chlorella pyrenoidosa that did not show significant alterations in cell density at concentrations of 0.1 and 1 mg/L during the first two days of exposure. However, at 10 mg/L there was a reduction in cell density. An increased cell density was observed after three days of exposure at 0.1, while 10 mg/L caused a decrease in cell density during all the five days of exposure. In addition, the authors also observed that the expression levels of ATP synthase and NADH dehydrogenase were significantly upregulated at 0.1 mg/L of BPA while at 10 mg/L the expression levels were significantly decreased [
42]. It has been demonstrated that bisphenols caused different effects during acute or chronic exposure tests. Indeed, in an acute test, 25 mg/L and 50 mg/L of BPA significantly inhibited the growth rate of both C
. pyrenoidosa and
S. obliquus during 96 hours of exposure, except for 24 h of exposure of
C. pyrenoidosa to 25 mg/L of BPA. On the contrary, both concentrations of 1 and 5 mg/L had a weak influence on the growth curve. During the chronic exposure, that lasted 30 days, BPA did not cause growth inhibition, while there was an increase in cell growth at both 1 and 50 mg/L of BPA with respect to the control [
43]. Lastly, in a 16-days experiment with the microalga
Stephanodiscus hantzschii treated with 0.01, 0.10, 1.00, 3.00, 5.00, 7.00 and 9.00 mg/L of BPA the authors observed that concentrations lower than 1 mg/L did not alter cell growth, while a concentration of 3 mg/L inhibited the algal growth during the first four days and then caused a growth stimulation. However, exposure to higher concentrations severely stopped algal growth and caused cell die when exposure time exceeded 8 days [
44].