1 Introduction

The contamination of aquatic environments by human activities concerns different study areas (e.g., social and environmental sciences, ecology, and economy), which demands an environmental health assessment to evaluate the effects on the aquatic biota (Corbi et al., 2008; Reddy & Behera, 2006; Schaider et al., 2019). Contaminants of emerging concern (CECs) are substances, synthetic or natural, found in personal hygiene products, medicines, and foodstuffs, among others, that reach water bodies through the incorrect disposal or domestic and industrial wastewater sewage (Hu et al., 2018; Luo et al., 2014; Sauvé & Desrosiers, 2014), and in this sense, both caffeine (CAF) and linear alkylbenzene sulfonate (LAS) are described as CEC (Brack et al., 2012; NORMAN, 2016).

CAF is the most widely consumed stimulant drug worldwide, commonly used as an active ingredient in commercial drugs to enhance mental and physical performance (Kolpin & Meyer, 2002; Snyder et al., 2007). This substance is also present in coffee, tobacco, spices, teas, sodas, and many other foods (Temple et al., 2017). However, CAF is not fully metabolized in the human body, and it can be eliminated in wastewater and reach the environment (Wang et al., 2011). Benotti and Brownawell (2009) observed that CAF half-life ranged from 3.5 to more than 100 days depending on the water’s physical and chemical characteristics. The continuous intake of CAF from anthropogenic sources raises its environmental concern (Daneshvar et al., 2012; Machado et al., 2016) that can be worsened by the increase of caffeine consumption in areas such as Europe (Quadra et al., 2020). LAS is a primary surfactant used in laundry detergent formulations worldwide, tending to be absorbed into suspended solids and accumulated in sediments (Zhu et al., 1998). Due to the amphiphilic nature of its carbon chain and its sulfonate group, this substance can be solubilized and easily transported by water (Hampel et al., 2012; Prats et al., 1993). Environmental contamination by LAS has been reported in many environmental compartments in concentrations from part per trillion to part per billion (Eichhorn et al., 2002; Sanderson et al., 2006) and the increase in the projected global market of surfactant demonstrates that the fate, distribution, and persistence of surfactants in the environment is an urgent matter (Badmus et al., 2021).

Both CAF and LAS are water quality indicators due to their constant presence in domestic wastewater, seawater, streams, groundwater, and the direct association with human activity (Brack et al., 2012; Buerge et al., 2006; Gonçalves et al., 2017; Moore et al., 2008; NORMAN, 2016). CAF was detected from 3.4 to 6.6 µg L−1 in effluents from wastewater treatment plants (WWTPs), reaching up to 127 µg L−1 in water bodies (Montagner & Jardim, 2011; Sui et al., 2010), while LAS occurred in WWTPs ranging from 0.4 to 2.5 mg L−1, and in streams with concentrations up to 1 mg L−1 (Eichhorn et al., 2002; Fox et al., 2000; Holt et al., 1998; Knepper et al., 2003). In this scenario, ecotoxicological assessment contributes to the environmental water quality monitoring, aiming to preserve the environment against adverse biological effects from multiple chemical contaminations (Altenburger et al., 2019; Hoogenboom et al., 1999).

The microcrustacean Daphnia magna Straus 1820 (Crustacea: Cladocera) has been used as a bioindicator since 1940 and stabilized as a standard organism-test in ecotoxicity assays in the 1970s (Anderson, 1944; Anderson & Jenkins, 1942). Offspring, juveniles, and adults compose the organism’s life cycle, and during the development, the organisms exchange their ecdysis (molting process), which allows them to grow and release their neonates. The molting process is controlled by the ecdysteroid hormones and can be influenced by CEC, including hormones and endocrine substances (Giraudo et al., 2017; Martin-Creuzburg et al., 2007; Sumiya et al., 2014).

The Daphnia magna reproduction test guideline is described by the OECD (2012) and considered as endpoints: the number of living offspring produced by each parent animal; the possible calculation of the concentration associated with x% of effect (ECx); the lower observed effect concentration (LOEC); and the no observed effect concentration (NOEC). Although these endpoints bring reference values for specific characteristics of the organism’s life cycle, there is a lack of qualitative indicators showing other chemical effects. During the Daphnia reproduction test, it is possible to record complementary parameters to the offspring number, including the time of the first molting, the frequency and number of molting throughout each organism’s life, and the association between molting and reproduction.

Although some CECs present low acute toxicity, these contaminants may cause significant reproductive effects even at low exposure levels (OECD, 2008). Nonetheless, these chemicals can have specific modes of action that may affect D. magna. Thus, analyzing qualitative endpoints can complement the investigation of toxic effects and evidence the D. magna life history strategy in response to environmental stressors. In this sense, we assessed the impact of CAF and LAS on the reproduction and molting processes of D. magna. Two scenarios were evaluated based on the (1) reported environmental concentrations and (2) expected effect concentrations. From the demand for further research on emerging pollutants, distinct approaches are fundamental to bring additional information regarding the organism’s responses to these pollutants. To evaluate this gap, we included qualitative indexes to the conventional quantitative endpoints.

2 Materials and Methods

2.1 D. magna Cultivation

According to the OECD Guidelines (OECD, 2012), daphnids used in the test were cultivated at the Aquatic Ecology Laboratory, at São Carlos School of Engineering, University of São Paulo, Brazil. The organism’s health status was assessed every 2 months with sensitivity bioassays using copper sulfate pentahydrate (CuSO4.5H2O, Synth®) as the reference substance (ABNT, 2016).

2.2 Exposure Design

The selected environmental pollutants were CAF (C8H10N4O2) (Sigma-Aldrich®, > 99% of purity) and LAS (C12H25C6H4SO3Na) (Sigma-Aldrich®, 80.7% of purity). The stock solutions (nominal concentrations) were prepared in distilled water (LAS 1.0 g L−1 and CAF 100 mg L−1). Two scenarios were proposed based on the literature: (1) reported environmental concentrations; (2) expected effect concentrations (Table 1).

Table 1 Reported environmental concentrations (scenario 1) and expected effect concentrations (scenario 2) selected for Daphnia magna reproduction tests
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The D. magna reproduction test was adapted from the OECD Guidelines (2012) and the test was carried out at a controlled temperature (20 ± 2 °C), and under 16 h light:8 h darkness. Reconstituted water was prepared as a mineral solution of CaCl2.2H2O (73.5 g L−1), KCl (5.8 g L−1), NaHCO3 (64.8 g L−1), and MgSO4.7H2O (123.3 g L−1) dissolved in distilled water under constant aeration (ABNT, 2016). The hardness (175–225 mg CaCO3 L−1) and the pH (from 7.6 to 8.0) were controlled. Daphnia magna was fed with yeast suspension (0.125 mg L−1), fish food (2.5 mg L−1), and microalgae (Raphidocelis subcapitata) grown in L.C. Oligo medium (3 × 105 cells mL−1) (ABNT, 2011).

A test control with only reconstituted water was used to evaluate the life cycle of D. magna without contamination. This test compared the daphnids’ ability to respond to environmental changes in the molting cycle and reproduction ratio. The reproduction test had 10 replicates that consisted of one neonate (< 24 h old) in a 100 mL beaker for each treatment (CAF and LAS concentrations, and control). The test was operated in semi-static conditions over 21 days, with the test solution’s renew and feeding three times a week in all replicates. The pH and conductivity of all treatments were measured weekly.

The parental immobility, occurrence of offspring, and molting were recorded daily. Throughout the assays, the offspring, molts, and immobile organisms were removed. Regarding the molting process from a quantitative approach, we analyzed CAF and LAS effects on the number of molts produced per parental D. magna and the number of molts produced until the primiparity stage (day of first brood release) and intermolt time. As outputs of the reproductive process, we analyzed the effects on offspring produced per parental D. magna, offspring produced per brooding (counting the first three broods in all CAF and LAS treatments and control), broods release time (the first day of exposure as starting time, with comparisons until the 4th brood for all treatments and control), and time between broods (considering the four first brood in all CAF and LAS treatments and control).

2.3 Statistical Analysis

The Shapiro-Wilk normality test (significance level p = 0.05) was applied to identify the type of data distribution. For data that showed normal distribution (p ≥ 0.05), the one-way ANOVA test was performed to investigate differences between CAF or LAS treatments and control. The Fisher’s test was applied to identify the specific differences pointed out by the previous test. Comparisons of independent samples by groups, such as scenarios 1 and 2 endpoints, were analyzed using the T-test. Regarding non-normal data analysis (p < 0.05), significant differences between CAF or LAS treatments and control were based on the Kruskal-Wallis test, followed by the two-tailed test. Moreover, comparisons of independent samples by groups were processed by the Mann-Whitney test. The statistical treatment of data was executed using Statistica® software version 7 and sensitivity bioassays by R® software version 3.3.5, packages MASS and DCR (R Core Team, 2018). Significant differences were considered when p ≤ 0.05, assuming a 95% confidence interval. The significant p-values are presented. The responses of the selected endpoints were plotted on graphs using OriginPro® software version 8.0.

2.4 Qualitative Indexes

We proposed additional endpoints on already established ecotoxicological assays to further comprehend the ability of D. magna to respond to a stressful environment. To evaluate these potential effects in the daphnids’ life cycle, we suggested calculating two indexes: molting effect index for the molting process (M) as %IM and offspring effect index for offspring production (O) as %IO index. The molts and the offspring were assessed daily for 21 days to calculate these parameters efficiently and minimize errors in the indexes. Furthermore, the cumulative number of molts corresponded to the measured value for each replicate ([M1, M2, …, Mn], where n = number of replicates) and the cumulative offspring produced for each replicate ([O1, O2, …, On], where n = number of replicates).

The last molt produced by a dead parental organism was used to calculate the offspring’s average or molt. If the parent died producing the sum of 3 molts and 15 offspring, this value was used to calculate the indexes on the 21st day, adding the mortality/immobility effect variation from the indexes’ replicates. The %IM (Eq. 1) and %IO (Eq. 2) indexes compare the effects of the environmental pollutants (CAF and LAS) and the test control calculated as an adaption of Sierra-Alvarez and Lettinga (1991) as follows:

$$\mathrm{\%}{\mathrm{I}}_{\mathrm{M}}=\left[\left({\mathrm{M}}_{\mathrm{EP}}-{\mathrm{M}}_{\mathrm{TC}}\right)/{\mathrm{M}}_{\mathrm{TC}}\right]\times 100$$
(1)

where:

M EP:

average number of molts measured over 21 days for each environmental pollutant concentration, average [M1, M2, …, Mn], n = 10 replicates;

M TC:

average number of molts measured over 21 days for each replicate of test control.

$$\mathrm{\%}{\mathrm{I}}_{\mathrm{O}}=\left[\left({\mathrm{O}}_{\mathrm{EP}}-{\mathrm{O}}_{\mathrm{TC}}\right)\right]\times 100$$
(2)

where:

O EP:

average of total offspring number measured over 21 days for each environmental pollutant concentration, average [O1, O2, …, On], n = 10 replicates;

O TC:

average of total offspring number measured over the 21 days of the assay for test control.

The effects of environmental pollutants on the molting process (%IM) and production of offspring (%IO) were summarized in five categories: “Induction,” “Evidence of induction,” “No effect,” “Evidence of inhibition,” and “Inhibition” (Table 2). The category “No effect” corresponds to 20% of the confidence range considering inadvertent parental mortality or accidental death (OECD, 2012) and standard deviation of offspring production between similar conditions (10 individual L−1, temperature between 20 and 25 °C) (Olkova et al., 2017).

Table 2 Classification of the effect of the molting process (%IM) and offspring production (%IO)
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3 Results

In general, throughout the D. magna reproduction test, the average pH ranged from 7.6 to 8.0. The average conductivity was 426.5, 435.2, and 509.0 µS cm−1 for the test control, CAF, and LAS treatments, respectively. The test control followed the OECD recommendations (OECD, 2012), with a minimum of 60 offspring production, immobility below 20%, and coefficient variation below 25% for offspring production. The D. magna’s health status was evaluated in sensitivity bioassays (CuSO4.5H2O), presenting EC50 of 0.0445 and 0.0453 mg L−1, comparable to values found in the literature (0.0546 mg L−1) that assure reproducible data (Struewing et al., 2015).

Parental Daphnia from CAF treatments scenario 1 and 20 and 40 mg L−1 in scenario 2 for CAF presented 20% of immobility or less, resulting in no negative effect over D. magna mobility. In contrast, immobility of 90% was recorded for 60 mg L−1 of CAF. Exposures to LAS showed that the immobility of parental Daphnia was dependent on the dose increase. LAS treatments from scenario 2 induced high immobility (90% at the 4.9 mg LAS L−1, and 80% at 4.1 mg LAS L−1). On the other hand, no immobility was recorded for LAS treatments from scenario 1 (from 0.4 to 1.0 mg LAS L−1).

3.1 Effects on Molting

3.1.1 Number of Molts Produced per Parental D. magna

The total number of molts produced over 21 days ranged from 75 in control, and among CAF treatments, 70 (0.005 mg L−1 of CAF) to 78 (0.127 mg L−1 of CAF) for scenario 1, and from 49 (60 mg L−1 of CAF) to 75 (20 mg L−1 of CAF). In LAS exposures, the total number of molts varied between 69 (1 mg L−1 of LAS) and 75 (0.4 mg L−1 of CAF), in scenario 1. For scenario 2, it ranged from 16 (4.9 mg L−1 of LAS) to 41 (4.1 mg L−1 of LAS). Fig. 1 shows the number of molts produced per parental Daphnia in 21 days according to exposure scenarios.

Fig. 1
figure 1

Number of molts produced per parental Daphnia magna over 21 days

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No statistical differences were identified between CAF treatments (scenario 1) and control, and between LAS treatments (scenario 1) and control in the number of molts produced by parental Daphnia throughout the exposures (p > 0.05, Kruskal-Wallis test). However, for expected effect concentrations (scenario 2), significant differences were observed comparing CAF treatments and control (p = 0.005) and LAS treatments and control (p = 0.0004), according to the Kruskal-Wallis test. The two-tailed test revealed that the number of molts in 60 mg L−1 of CAF (5 ± 2 molts) and 4.9 mg L−1 of LAS (2 ± 2 molts) were statistically different from the control (8 ± 1 molts) (CAF: p = 0.008 and LAS: p = 0.0002, respectively). Comparing the number of molts produced per parental Daphnia between scenarios, the Mann-Whitney test pointed out no differences between CAF treatments scenarios 1 and 2 (p > 0.05), in contrast to that observed between LAS treatments scenarios 1 and 2, which showed a significant difference (p = 0.000003).

3.1.2 Molting Process Until Primiparity

Except for the 0.005 mg L−1 of CAF, 2.5 mg L−1 of LAS, and 4.9 mg L−1 of LAS treatments, parental Daphnia reached primiparity by producing an average of 5 molting (Fig. 2). There were no significant differences between CAF or LAS treatments and control, considering scenarios 1 and 2 (p > 0.05, Kruskal-Wallis test). Besides, comparing the number of molts until the first reproduction between CAF treatments from scenarios 1 and 2, and LAS treatments from scenarios 1 and 2, no statistical differences were identified (p > 0.05, Mann-Whitney test).

Fig. 2
figure 2

Number of molts until D. magna primiparity

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3.1.3 Intermolt Time

In general, the average intermolt time varied from 2 to 3 days in control, CAF, and LAS treatments. Fig. 3 shows the intermolt time of parental Daphnia in the experiments. According to the Kruskal-Wallis test, statistical differences were pointed out between CAF treatments (scenario 2) and control (p = 0.03), and the 60 mg L−1 of CAF treatment showed a significant difference compared to the control (p = 0.02, two-tailed test). Statistical similarities were observed between CAF treatments (scenario 1) and control, and between LAS treatments (scenarios 1 and 2) and control (p > 0.05, Kruskal-Wallis test). Comparing the intermolt time between CAF treatments from scenarios 1 and 2, and LAS treatments from scenarios 1 and 2, no significant differences were found (p > 0.05, Mann-Whitney test).

Fig. 3
figure 3

D. magna intermolt time

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3.1.4 Molting Effect Index

From the %IM data, only the 60 mg L−1 of CAF treatment presented a negative response to environmental changes in the molting cycle ratio, classified as “evidence of inhibition” (Table 3). The 0.005 to 40 mg L−1 of CAF treatments did not affect the molting process (“no effect”). Besides, the %IM of LAS showed concentration-dependent effects on the D. magna life molting cycle. There was no evidence of induction or inhibition for 0.4 to 2.5 mg LAS L−1 treatments, classified as “no effect.” Nevertheless, higher concentrations of LAS produced inhibition response, specifically for 4.1 and 4.5 mg LAS L−1, which resulted in “evidence of inhibition” classification, and for 4.9 mg L−1, where an “inhibition” effect was determined.

Table 3 Molting effect index (%IM) calculated for CAF and LAS exposures in scenarios 1 and 2
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3.2 Effects on Reproduction

3.2.1 Offspring Produced per Parental D. magna

Over 21 days, parental daphnids from the control test produced a total of 709 offspring. For CAF reported environmental concentrations (scenario 1), the total offspring varied between 603 (0.0005 mg L−1) and 760 (0.127 mg L−1). In the CAF scenario 2 (expected effect concentrations), the range of total offspring produced was between 199 (60 mg L−1) and 732 (40 mg L−1). In LAS treatments, the total offspring produced ranged from 865 (1 mg L−1) to 963 (2.5 mg L−1) for scenario 1, and from 67 (4.9 mg L−1) to 191 (4.5 mg L−1) for scenario 2. Fig. 4 shows the number of offspring produced per parental Daphnia in 21 days according to exposure scenarios.

Fig. 4
figure 4

Number of offspring produced per parental D. magna over 21 days

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No statistical differences were observed in the number of offspring produced by parental Daphnia between CAF treatments and control for scenario 1 (p > 0.05, Kruskal-Wallis test). For scenario 2, differences between CAF treatment and control were pointed out (p = 0.0005, Kruskal-Wallis test). According to the two-tailed test, the number of offspring produced by parental Daphnia in the 60 mgL−1 of CAF treatment showed a statistical difference with the control (p = 0.01). Moreover, no statistical difference was observed in the number of offspring produced between scenarios 1 and 2 in CAF exposures (p > 0.05, Mann-Whitney test).

Statistical differences were observed between LAS treatments and control for scenario 1 (p = 0.02, Kruskal-Wallis test), and this difference occurred between 2.5 mg L−1 and control (p = 0.01, two-tailed test). For LAS scenario 2, statistical differences were identified between LAS treatments and control (p = 0.0001, Kruskal-Wallis test). The 4.1, 4.5, and 4.9 mg L−1 treatments showed statistical difference with control (p = 0.008, p = 0.01, and p = 0.001, respectively), according to the two-tailed test. The number of offspring produced in scenario 1 was statistically different from scenario 2, in LAS exposures (p < 0.05, Mann-Whitney test).

3.2.2 Offspring Produced per Brooding

Regarding the number of broods, the first four broods were registered in the control test, 40, 60 mg L−1 of CAF, and 1, 2.5, 4.1, 4.5, and 4.9 mg L−1 of LAS. In the treatments 0.005, 0.03, 0.127, and 20 mg L−1 of CAF and 0.4 mg L−1 of LAS, parent daphnids reached 5 broods. Fig. 5 shows the number of offspring per brood in CAF (a) and LAS (b) exposures. In the 1st brood, there were no significant differences in the number of offspring between CAF treatments (scenario 1) and the control, and CAF treatments (scenario 2) and control (p > 0.5, according to the one-way ANOVA test). Statistical differences between LAS treatments (scenario 1) and control were pointed out (p = 0.03, one-way ANOVA test). According to Fisher’s test, there are differences in the number of offspring produced per parental Daphnia between 1, 2.5 mg L−1 of LAS, and control (p = 0.02 and p = 0.01, respectively), in which there was an increase in the average number of offspring from the 1st brood (33 to 40%) compared to the control. In contrast, there were no differences between LAS treatments (scenario 2) and control (p > 0.05, one-way ANOVA test).

Fig. 5
figure 5

Number of offspring per daphnids brood: a CAF treatments; b LAS treatments

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In the 2nd brood, statistical differences were observed between CAF treatments and control for both scenarios (p = 0.02, one-way ANOVA test). Fisher’s test pointed out differences between 0.005, 0.03, and 0.127 mg L−1 of CAF and control (p = 0.003; p = 0.04; and p = 0.04, respectively) for scenario 1, showing a decrease in the average number of offspring (17 to 30%), compared to the control. Differences between 60 mg L−1 of CAF and control (p = 0.01) for scenario 2 were also identified on Fisher’s test (39% of offspring decrease in this CAF treatment). Similarly, there are differences between LAS treatments and control for scenarios 1 and 2 (p = 0.0001 and p = 0.02, respectively, one-way ANOVA test). According to the Fisher’s test, the number of offspring produced in 1 and 2.5 mg L−1 of LAS treatments (scenario 1), and 4.1, 4.5, and 4.9 mg L−1 of LAS treatments (scenario 2) showed statistical difference with control (p = 0.02, p = 0.000008, p = 0.04, p = 0.03, and p = 0.02, respectively). For these statistically significant treatments, an increase in the average number of offspring from 22 to 52% (LAS treatments, scenario 1) and a decrease of 26 to 48% (LAS treatments, scenario 2) were observed.

Regarding the 3rd brood, the number of offspring, whether produced in the CAF or LAS treatments, showed no significant differences with the control for both scenarios (p > 0.5, one-way ANOVA test). Comparing the number of offspring produced per parental Daphnia between scenarios in each brood posture (T-test), statistical differences were identified between CAF treatments of scenarios 1 and 2, and LAS treatments of scenarios 1 and 2 for 1st brood (CAF: p = 0.004 and LAS: p = 0.01), and 2nd brood (CAF: p = 0.006 and LAS: p = 0.000003), respectively. Nevertheless, there were no differences between scenarios considering the number of offspring from the 3rd broods of CAF and LAS treatments (p > 0.05), according to the T-test.

3.2.3 Broods Release Time

Fig. 6 shows the brood release time of parental Daphnia in CAF and LAS exposures. Significant differences were observed between CAF treatments of scenario 1 and the control, for the 1st (p = 0.03), 2nd (p = 0.01), 3rd (p = 0.003), and 4th (p = 0.03) broods, according to the Kruskal-Wallis test. The two-tailed test pointed out that the release time for the 3rd brood in 0.005 mg L−1 of CAF treatment differs from the time recorded in control (p = 0.04), highlighting an average reduction of 2 days of the offspring’s posture. We observed anticipation of the brood release (an average of 1 to 3 days, compared to the control) for 0.127 mg L−1 of CAF in the first four broods (1st brood, p = 0.05; 2nd brood, p = 0.02; 3rd brood, p = 0.004; 4th brood, p = 0.02), according to the two-tailed test. In CAF treatments scenario 2, and LAS treatments scenarios 1 and 2, no statistical differences were identified comparing each brood’s release time and the control (p > 0.05, Kruskal-Wallis test). The Mann-Whitney test showed that there are significant differences in brood release time of parental Daphnia from CAF treatments between scenarios 1 and 2 for the 1st (p = 0.00005), 2nd (p = 0.00003), and 3rd (p = 0.005) broods. For the 4th brood of CAF and LAS treatments, no significant differences were identified between scenarios 1 and 2 (p > 0.05, Mann-Whitney test).

Fig. 6
figure 6

Brood release time: a CAF treatments; b LAS treatments

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3.2.4 Time Between Broods

The elapsed time between 1st–2nd broods, 2nd–3rd broods, and 3rd–4th broods is presented in Fig. 7. Significant differences between treatments and control were only for LAS scenario 1 (p = 0.009, Kruskal-Wallis). The elapsed time between 2nd and 3rd broods in 1 mg LAS L−1 showed significant differences with the control (on average 1 day less than the standard registered in the control), p = 0.02, two-tailed test. There were no significant differences between treatments and control for CAF scenarios 1 and 2, and LAS scenario 2 (p > 0.05. Kruskal-Wallis). According to the Mann-Whitney test, comparing CAF treatments scenarios 1 and 2, only the time between 1st and 2nd broods was statistically different (p = 0.001). No significant differences compared the time considering LAS treatments scenarios 1 and 2 (p > 0.05, Mann-Whitney test).

Fig. 7
figure 7

Time between broods: a CAF treatments; b LAS treatments

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3.2.5 Offspring Effect Index

Different results were found by the %IO (Table 4), no difference between CAF in scenario 1 and control was observed, and “no effect” classification was recorded. In scenario 2, with CAF exposures, the %IO presented effect only with 60 mg L−1 of CAF, classified in the category “inhibition.” On the other hand, an induction effect was evidenced for LAS scenario 1, classified as “evidence of induction.” Adversely, scenario 2 with LAS presented an “inhibition” effect.

Table 4 Offspring effect index (%IO) calculated for CAF and LAS exposures in scenarios 1 and 2
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4 Discussion

Daphnia’s EC50 for short-time exposure (48 h and 96 h) with CAF in the literature ranges from 177.8 to 445.3 mg L−1 of CAF (Chevalier et al., 2015; Di Lorenzo et al., 2019; Lilius et al., 1995; OECD SIDS, 2004). Although CAF is considered a low-risk CEC to aquatic biota, the mixture with other compounds combined with the bioaccumulation capacity in some aquatic species brings attention to the necessity of detailed analysis of its potential environmental hazard (Beasley et al., 2015; Dafouz et al., 2018; Palma et al., 2018). The LAS effect on mobility for D. magna acute exposure varies from 0.26 to 55 mg LAS L−1 (OECD SIDS, 2005) and depends on the number of carbons in LAS composition and homologs distribution (Prats et al., 1993; van de Plassche et al., 1999; Verge et al., 2001). In our paper, the LAS’ negative effect over mobility in the long-term exposure was observed only for scenario 2. The study from van de Plassche et al. (1999) calculated the geometric mean normalized of long-term NOEC for C11.6 LAS in 12 samples and obtained a concentration of 1.4 mg L−1 for D. magna, explaining why no effect over immobility was observed in scenario 1 for LAS in our study.

Despite recent and constant publications on D. magna ecotoxicity tests, few studies have evaluated the CAF and LAS chronic exposure. On the other hand, although our immobility endpoints demonstrate the effects on D. magna for the highest concentrations (scenario 2 for LAS and 60 mg L−1 of CAF), the reported environmental concentrations (scenario 1) and some tested scenario 2 of CAF (20 and 40 mg L−1) did not present any effect on D. magna responses with immobility. Thus, comprehending the effects beyond the classic toxicity endpoints can provide additional support for long-term toxicity studies using LAS and CAF as contaminants (Lewis, 1991).

4.1 Effects on Molting

The scenario 1 for CAF and LAS resulted in no effect on molts produced per parent. However, in scenario 2, the highest CAF concentration presented lower molts produced per parental Daphnia and slower intermolt time, resulting in “evidence of inhibition” for %IM. Besides, the highest LAS concentration in scenario 2 significantly reduced the number of molts and produced an “inhibition” effect for %IM. Bang et al. (2015) evaluated the D. magna survival and reproduction over 21-day exposure to CAF, and no difference in average body length was evidenced for 8.9 mg L−1 of CAF compared to the control. Our results demonstrated a decrease in molting numbers for 60 mg L−1 of CAF (scenario 2) by %IM that can be related to possible inhibition of growth, as seen by Lu et al. (2013) for 10 mg L−1 of CAF. These results indicate that the effect over Daphnia’s growth is concentration-dependent, reinforced by the slower intermolt time recorded for this CAF concentration, classified as expected effect scenario.

We observed that increasing CAF concentrations led to an improper molting process, since after the release, the old exoskeleton was still attached to the organism’s body, affecting D. magna’s swimming capability. This release at the end of the molting cycle depends on different enzymes and inorganic ions (Duchet et al., 2011; Reynolds & Samuels, 1996), and the incomplete exoskeleton separation can lead to molting disruption and, in the worst case, organism’s death (Song et al., 2017). Few changes in the molting process may not influence overall molting frequency, but, even then, the dysfunctional process confirms that the organism’s life is being affected by the toxic compound.

In our study, only 4.9 mg L−1 of LAS in scenario 2 presented a statistically distinct effect on molts produced per parent and intermolt time to the control, but with %IM, a significant variation of molt production was evidenced for all LAS concentrations from scenario 2, showing an inhibition effect. As some statistics could generate false evidence of a biological growth (Lytle, 2001), our result on the molting process endorses the necessity of distinct approaches easily adopted from classical reproduction toxicity tests to detail the ability of D. magna confronted by a stressor on the environment. LAS’s concentrations with “no effect” classification in %IM demonstrated that the difference between average number of molts for the test control and scenario 1 can be assumed as a natural deviation. In this sense, this index confidence range demonstrates lower misestimation of D. magna behavior and could reduce false positive or negative effects results when undertaking ecotoxicological evaluation. As any disturbance in the normal molting cycle might increase the Cladocera’s sensitivity to the toxicant (Bodar et al., 1990), the inhibition effect in %IM was evidenced for concentrations with immobility higher than 80%. Alterations in the molting process can indicate disruption of a multi hormonal system controlled by ecdysteroids (Chang, 1993; Giraudo et al., 2017; LeBlanc, 2007; Mu & LeBlanc, 2002). As no effect was observed in the molts until primiparity for both scenarios with CAF and LAS, we can indicate that the toxic effect over molting needs time to be evidenced.

4.2 Effects on Reproduction

The presence of chemicals in the aquatic environment can affect D. magna’s brood quantity and quality (Campos et al., 2016). Overall, the number of offspring produced per parent was more affected by scenario 2 than scenario 1 for both CAF and LAS, which was expected as scenario 2 was described as expected effect concentrations. An %IO inhibition effect was recorded for 60 mg L−1 of CAF and for all LAS concentrations from scenario 2. Lu et al. (2013) registered a decreasing offspring number per brood with a CAF increase, and with 10 mg L−1 of CAF, they observed a 66% reduction in offspring produced per brood per female compared to control. In scenario 1, only 2.5 mg L−1 of LAS affected the offspring production, resulting in a higher number than the control. Differences in offspring production may be related not only to the type of contaminant but also to its concentration and increase in the offspring produced per parent causes changes in the life cycle of D. magna and can impact the dynamics of aquatic ecosystems, producing a negative effect on the population (Cleuvers et al., 1997; Goser & Ratte, 1994; Preuss et al., 2009). In our experiments, the results suggested that environmental concentrations of CAF did not increase the offspring produced but induced a fast brood release.

The offspring produced depended directly on LAS concentration and presented two distinct effects, an %IO induction was evidenced for scenario 1 concentrations and %IO inhibition occurred in scenario 2 concentration. A previous study has indicated that LAS presents low hazard effects under detected environmental concentrations, presenting chronic NOECs from 1.2 to 3.2 mg L−1 (Taylor, 1985). The results found in this paper indicated that the environmental contamination of LAS could increase D. magna population. However, this could lead to an overcrowding effect, negatively impacting aquatic ecosystem dynamics over the population (Cleuvers et al., 1997; Goser & Ratte, 1994; Preuss et al., 2009). Our data demonstrate the necessity of analyzing different effects of ecological importance in classic toxicity tests, such as alteration of reproductive strategy and change in growth rate (Hayes et al., 2002). Ecologically, the presence of a chemical can affect both the quantity and quality of the offspring produced (Campos et al., 2016), and through %IO index, we evidenced the distinct effect of LAS over the quantity of offspring produced. Therefore, these differences are not only dependent on the chemical type but vary with LAS concentrations.

4.3 Relation Between Molts Number and Offspring Production

During the organism’s natural life cycle, neonates are released during ecdysis and, after the molting, another set of oocytes begins to move to the maturation phase in the incubator chamber; therefore, it would be expected that the acceleration in the ecdysis process is accompanied by the induction of neonates production (Dodson et al., 2010). However, in our study, there was no evidence of molting induction when offspring induction was recorded (scenario 1 for LAS). We can suggest that, from the relationship between the rate of neonates’ production and the process of ecdysis, the organism allocates energy primarily for the species’ perpetuation (Dodson et al., 2010).

Different pollutants show contrasting dominant ecotoxicological modes of action (Barata & Baird, 2000), and from the %IM and %IO indexes, it is possible to compare different reproductive strategies of D. magna. In scenario 1, daphnids did not alter their molt production, but increased their offspring with LAS. Environmental contamination can result in changes in daphnids’ density, growth, and reproduction. As this species feeds on primary producers (e.g., algae) and serves as food for final consumers (e.g., fish), these changes may cause disequilibrium in this population level and consequently in the food chain (Tanaka et al., 2018), highlighting the importance of studying the individual’s growth and reproduction effects (Bruijning et al., 2018). Moreover, with the constant increase usages of the CAF and LAS worldwide (Badmus et al., 2021; Quadra et al., 2020), its environmental concentrations are expected to increase, and scenario 2 could represent real scenario, demonstrating how important is to evaluate these scenarios in our paper.

The complementary indexes proposed in this paper (%IM and %IO) were suitable for evaluating induction and inhibition effects with LAS and CAF and can complement D. magna classic toxicological analysis for lethal and sublethal conditions. Moreover, the main advantage of applying these suggested indexes is to deduce any distinct behavior by observing the organism’s development. The daily analysis allows the verification of the swimming trend and how the organism uses its energy (e.g., growth or reproduction), and complements the indexes’ interpretation.

5 Conclusion

LAS and CAF were used in this paper for analyzing and comprehending the toxic effects on molting and reproduction process in long-term exposures. According to our findings, the indexes %IM and %IO were comparable to other endpoints in two scenarios and can add information on D. magna ecotoxicological analysis. In the same assay, we have results of classical endpoints, and we can qualify the information about the molting and reproduction process by calculating the %IM and %IO indexes. From the results, we can indicate that LAS and CAF produced contrasting dominant ecotoxicological responses. From the %IM and %IO data, it is possible to verify that LAS produces no effect on the molting process and an “induction” effect on offspring production in reported environmental concentrations (scenario 1). Moreover, in scenario 2, LAS produced an “inhibition” effect over %IO and “evidence of inhibition” and an “inhibition” effect on the D. magna molting production. CAF results indicated that in expected environmental concentrations, scenario 1, no effect was observed in both indexes. Our results indicated that CAF inhibited more offspring than molting for 60 mg L−1 of CAF. This complementary information on the Daphnia life cycle with LAS and CAF demonstrated this approach to classic toxicological data for environmentally relevant pollutants. Further use of these indexes with distinct substances can improve information on ecotoxicological assessments.