Journal Pre-proof Daphnia magna responses to fish kairomone and chlorpromazine exposures

27 To avoid being preyed, organisms must be able to identify predatory threats by sensing 28 molecules released by predators (kairomones), and to employ effective strategies to 29 avoid detection by predators. Furthermore, in the wild, organisms are also exposed to 30 chemicals that may alter their behavioral traits, such as neuroactive pharmaceuticals. 31 Considering the co-occurrence of both types of chemicals, their possible interaction 32 needs to be studied. To address this topic, the aim of this study was to verify the effects 33 of fish kairomone (FK – a chemical associated to putative predation by fish) and 34 chlorpromazine (CPZ - neuroactive pharmaceutical drug, environmental contaminant), 35 isolated and in combination, in different functional endpoints of Daphnia magna , such 36 as oxygen consumption, feeding rate, behavior and reproduction. Among these 37 endpoints, oxygen consumption was only affected by the combination of compounds 38 (FK + CPZ). On the other hand, feeding rate was affected by all treatments, being 39 smaller than control. For life history traits and phototactic behavior, the effects of FK 40 predominated over the ones caused by CPZ exposure, incrementing the reproductive 41 output of females, leading to greater population growth rates and decreasing swimming 42 behaviour. 43


Chlorpromazine Fish kaironome -Leuciscus idus
To obtain fish kairomone (FK), two 8 cm juvenile fish (Leuciscus idus) were allowed to 146 swim in a12 L aquarium, filled with ASTM hard water medium for 24 h, after which the 147 medium was filtered (0.045 µm). This procedure was repeated for every experiment, to 148 use a fresh FK solution for every experiment. Only for the reproduction tests, a single 149 FK preparation was used in three media changes. This method is routinely used to consumption tests, to allow the possibility of comparing data from all these endpoints, 219 and, in our laboratory conditions, 15 d old organisms have an average size of 2.76 ± 0.2 220 mm (N = 56, Mean ± SD).

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Reproduction assay 222 Reproduction test was conducted according to OECD recommendations (OECD, 2012).

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Organisms less than 24-h old were exposed, individually, in 100 mL glass flasks, to 224 each experimental condition and a total of 10 replicates per condition was used.

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Obtained results indicated that the tested compound was stable in ASTM water since were similar to those aged for 24 (1073.5 ± 5.6 µg/L) and 48 h (1063.2 ± 11.5 µg/L).

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The oxygen consumption was smaller (F 3;12 = 4.091, p<0.05) than control in the less studied, and this study brings some perspectives about this potential association.

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The here-adopted strategy is a comprehensive framework that includes a series of 282 multiple endpoints with high physiological importance, whose impairment may result in 283 strong population effects.

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As a sensitive metabolic biomarker and a high potential for toxicity screening, smaller when the organisms were exposed to the mixture, that is, less oxygen was 291 consumed. This can be an indication that anthropogenic and natural stressors can 292 interact and cause metabolic alterations in exposed animals. Although not significantly, 293 oxygen consumption in CPZ and FK alone were smaller than control, thus it is possible 294 to hypothesize that the joint effect of both stressors was additive. Concomitant with the 295 reported oxygen effects, females exposed to the combination of FK and CPZ also 296 moved less, thus, requiring lower oxygen consumption. In addition, another explanation 297 for the here observed effects is the relationship between oxygen consumption and 298 distance moved; both analyzed endpoints suffered a decrease after exposure to the 299 combination FK+CPZ. Thus, these two effects possibly indicate that impaired oxygen 300 consumption is associated with shorter distance moved, i.e. reduced swimming activity.

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In metabolic or respiration rate, food ingestion is an energetically expensive action and • Feeding rate was decreased in all treatment conditions; • Oxygen consumption was only affected in the mixture treatment (CPZ + FK); • For life history traits and phototactic behavior the effects of FK dominated those of CPZ

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: