3.1 Reproduction test conditions
Evaluating the best configuration for chronic exposure in preliminary tests, we observed that in 4 days, the number of new organisms did not exceed 5 in any of the aeration conditions, while in the other exposure times (7d and 10d) each organism had fission at least once. During the exposure time of 7d, the mean number of new organisms was 31 and 36 in the absence and presence of aeration, respectively. A high number of new organisms in the presence of aeration was not observed in the exposure time of 10d, in which the mean number of new organisms at the replicates without aeration was 67 against 52 in the replicates with aeration (Fig. 1). No statistical differences in the number of new organisms were pointed out comparing the presence or absence of aeration (Two-way ANOVA, p-value of 0.471). On the other hand, focusing on the exposure time, the total offspring in 4d showed a significant difference compared to 7d (p-value of 7.68 10−5) and 10d (p-value of 1.19 10−8).
According to Smith et al. (1991), species from the Naididae family present new generations of individuals within a period of 3 to 7 days. Özpolat et al. (2016) state that the P. longiseta (as P. leidyi) species takes 4 to 6 days to perform body regeneration after reproduction by paratomic fission. However, the authors also state that it is possible for multiple fission zones to form after the initial fission, whereby the organism is able to divide into more than 2 individuals. In this context, due to a good response in the simpler configuration and short time, we decided to continue the evaluation of P. longiseta in reproduction studies following the configuration without aeration and exposure time for 7 days, aiming to obtain the first generation.
We emphasize that the suggested duration for short-term exposure (acute testing) is 48 hours for P. longiseta (Smith et al. 1991; Castro et al. 2020b). Another preliminary investigation of the exposure time of 4 d was reported by Castro et al. (2020b); they observed the presence of new individuals after 72 h (3 days) testing the configuration of acute toxicity bioassay. The duration of chronic assays applied to other organisms was also considered, such as those performed for Chironomus sp. (10d), an aquatic invertebrate (OECD 2011), the tropical aquatic oligochaete Allonais inaequalis (10d) (Corbi et al. 2015; Felipe et al. 2020). Determining the configuration of chronic tests lasting 7 days, without aeration, we obtained a fast response in chronic tests for aquatic invertebrates, reducing time and costs in the application of the tests.
3.2 Ecotoxicological assessment
The reproduction bioassay (7d without aeration) was successfully applied for three environmental contaminants and the reference substance. The reference substance (KCl) caused a constant decrease in the number of new organisms according to the concentration increase (Fig. 2a). Besides, the TBBPA at the lowest concentration induced a significant decrease in reproduction (mean of 3 new organisms at 300 µg/L) (Fig. 2b). On the other hand, the low concentration of SMX and low percentual dilution of sugarcane vinasse induced a reproduction rate near the control, and a sharp drop was observed at 86 µg SMX/L (no new organism; Fig. 2c) and at 4.95% of vinasse (mean of 2 new organisms; Fig. 2d).
According to the Kruskal-Wallis test, significant differences were identified between the reproduction of organisms exposed to doses of all contaminants or reference substances, and the control (p ≤ 0.05). Dunn's post hoc test showed that the reproduction of P. longiseta at 450, 675, 1000 and 1500 µg/L of the TBBPA was significantly different from the reproduction registered in the control. For SMX, the concentrations that showed a significant difference compared to the control were 86, 128, and 192 µg/L. For sugarcane vinasse, only the 7.4% dilution showed a significant difference to the control, and for the KCl, the concentrations with a significant difference in the organism's reproduction were 0.7, 0.9, and 1.3. Thus, only at the lowest concentrations and dilutions of the samples were no statistically toxic effects identified comparing the results to control samples (TBBPA: 300 mg/L and methanol control; SMX: 38, 55 mg/L, and methanol control; KCl: 0.5, 0.3 mg/L; sugarcane vinasse: 1.5, 2.2 and 3.3%) Moreover, the classic ecotoxicological parameters EC50, NOEC, and EC10 were obtained. Among the assessed substances, sulfamethoxazole showed the highest toxicity, presenting an EC50 of 59.9 µg/L, followed by Tetrabromobisphenol-A (EC50 =166.1 µg/L). The sugarcane vinasse caused an EC50 of 4.26%, indicating that this dilution caused an inhibitory effect of 50% on reproduction. Besides, the reference substance, KCl indicated an EC50 of 0.51 g/L (Table 1).
Table 1
Ecotoxicological endpoints obtained after chronic exposures of Pristina longiseta to KCl, TBBPA, SMX and sugarcane vinasse, expressed as EC10, EC50, and NOEC
Contaminants
|
EC50
|
Standard error
|
NOEC
|
EC10
|
KCl
|
0.51
|
0.03
|
0.50
|
0.12
|
TBBPA
|
166.1
|
0.70
|
300
|
5.56
|
SMX
|
59.9
|
1.83
|
55
|
52.1
|
Sugarcane vinasse
|
4.26
|
0.23
|
4.95
|
3.25
|
Values in g/L for KCl; in µg/L for tetrabromobisphenol-A and sulfamethoxazole; and percentage of dilution for sugarcane vinasse. |
By assessing EC10, we verified that the substances did not follow the same pattern as the toxicity of the EC50. SMX caused an EC10 of 52.1 µg/L, a concentration detected that poses a risk to the organisms, with a value close to the EC50. The effect observed in 10% of the organisms for tests with TBBPA was 5.56 µg/L, indicating that the contaminant shows effects at concentrations much lower than the identified EC50. Comparing the effects of SMX and TBBPA, we observed that SMX was more toxic (EC50 59.9 µg/L) than TBBPA. However, TBBPA causes a toxicity effect in 10% of organisms (EC10 5.56 µg/L) at much lower concentrations than SMX, showing that the species of P. longiseta was more sensitive to the antibiotic due to a window between the effect observed in 10 and 50% of the organisms to be smaller when compared to that observed in TBBPA. KCl caused a LOEC of 0.12 g/L, and raw sugarcane vinasse also had the unobserved effect near the EC50, at 3.25%.
Pristina longiseta is known to be more sensitive to the reference substance KCl in acute bioassays when compared to Allonais inaequalis, another native Brazilian Oligochaeta. Castro et al. 2020a observed a LC50 of 1.36 g/L for the short exposure of P. longiseta to KCl, whereas Corbi et al. (2015) found a LC50 of 3.5 g/L for A. inaequalis. The same was observed in chronic bioassays, where the EC50 for P. longiseta was close to the EC10 found by Felipe et. al (2020) for A. inaequalis (0.50 g /L), in 10-day chronic bioassays. The concentration of the effect on 50% of P. longiseta offspring (EC50 0.51) is a concentration of the beginning toxic effect in A. inaequalis.
Regarding the flame retardant, for the crustacean Daphnia magna, Yang et al. (2012) observed that TBBPA changed the reproduction rate of the individuals, presenting EC10-21d of time to the first brood, the total number of spawning and number of broods of 84 µg/L; 16 µg/L and 139 µg/L. Showing that for the observation of the effect in 10% of organisms, P. longiseta (EC10 5.56 µg/L) was more sensitive to exposures to TBBPA than the microcrustacean Daphnia magna. Pittinger and Pecquet (2018) reported that the effect of TBBPA on the reproduction of D. magna expressed as NOEC was above 300 µg/L, a value in agreement with that found in this research. Moreover, studies using the marine mussel Mytilus galloprovincialis showed that TBBPA induced the development of gametes in female and male individuals at concentrations below 375 µg/L (Wang et al. 2021). In addition, other authors point to TBBPA as an endocrine-disrupting agent in aquatic invertebrates, causing, in addition to impacts on reproduction, effects on species development (Yang et al. 2012; Pittinger and Pecquet, 2018; Wang et al. 2021). Corroborating the authors, our results indicated that this substance presents negative effects on the reproduction rate from concentrations below 200 µg/L. The review of the presence of TBBPA in different experiments showed that in freshwater environments it remains below 4.8 µg/L and in sediment samples below 480 ng/g dw, but in industrial and e-waste areas these values can be higher (e.g., 9750 ng/g dw) (Liu et al 2016). Moreover, it is known that this compound has the capacity to accumulate in different tissues of aquatic biota (Harrad et al 2009; Gong et al. 2021) and was a concern regarding its effects in long exposure.
For SMX, P. longiseta was more sensitive (EC10 of 52.1 µg/L) when compared to the alga Pseudokirchneriella subcapitata (EC10 of 150 µg/L); the microcrustacean Ceriodaphnia dubia (EC10 of 250 µg/L) and the cnidarian Hydra attenuata (EC10 of 5000 µg/L) (Straub, 2015). Comparatively, the reproduction of the naidid P. longiseta was more sensitive to SMX when compared to the species studied by Straub et al. (2015), which may be an indication that the reproduction of species of the Oligochaeta class is more susceptible to inhibition when exposed to antibiotic SMX in an environmentally relevant concentration. Qiu et al (2020), found that exposure to SMX has chronic and sub chronic effects on Danio rerio zebrafish, delaying egg hatching and impacting fish body size. In addition, other studies have also observed the effects of oxidative stress in the microalgae Raphidocelis subcapitata (Zhang et al. 2021), and inflammatory effects on fish, Ctenopharyngodon idella (Wang et al. 2021), Oreochromis niloticus (Hu et al. 2021) and Danio Rerio (Qiu et al. 2020). Studies show that at relevant environmental concentrations, SMX can cause chronic effects in different aquatic organisms, corroborating the results observed for P. longiseta in this study. In this context, the need to investigate the effects of these contaminants on other tropical aquatic worms is evident, as the bibliography for these organisms is scarce.
Due to the potential toxicity of sugarcane vinasse and its negative effects on aquatic biota (Silva et al. 2007; Christofoletti et al. 2013), in the 1970s, restrictive laws were established prohibiting the vinasse disposal directly or indirectly in water bodies (Fuess; Garcia, 2014; Moraes et al. 2015). Sugarcane vinasse is commonly applied in cane cultivation as fertigation and most ecotoxicological studies related to vinasse are carried out using soil organisms (Pedrosa et al. 2005; Coelho et al. 2017; Vilar et al. 2018; Sousa et al. 2019, Felipe et al. 2021). Therefore, there is a body of literature lacking answers to the vinasse effects (acute and chronic) on aquatic worms from tropical regions. Verma and Dalela (1976) performed toxicity tests with two species of fish, Puntius sophore and Mystus vittatus; they observed that 6.3% to 10% of vinasse caused mortality in 50% of these organisms after 96h of exposure at 32 ± 2ºC. Moreover, an increase in mucus production and a reduction of proteins in liver, brain, kidneys, and muscles of Channa punctatus were reported in dilutions from 50% of vinasse (Kumar and Gopal, 2001). Regarding chronic studies, two species of aquatic insect had the reproduction analyzed. For Drosophila melanogaster it was observed that 25% of vinasse decreases the egg fertility rate, and for Chironomus sp., 6.5% affects the emergence rate (Yesilada, 1999; Nyakeya et al. 2018). The P. longiseta reproduction test showed that 4% of nature vinasse can affect 50% of the offspring, showing again the high sensitivity level of this freshwater worm species.
There is a need for protocols that evaluate the potential effects of contaminants on freshwater Oligochaeta species. The fact that Pristina longiseta shows effects at concentrations below those of other aquatic organisms is a good indication that P. longiseta can be used to assess environmental contamination. As it is a benthic organism, P. longiseta can be an excellent indicator of contaminants not only in the water column but also into the sediment, even at low concentrations in the medium. In general, our results showed that the chronic test protocol can respond to the effects of chemical and environmental samples on the reproduction of P. longiseta.