Precopulatory Pairs Research Articles

Microplastics impair the reproductive behavior and life history traits of the amphipod Parhyale hawaiensis

We investigated the distribution and effects of waterborne microplastic (MP) (polyethylene microspheres, 53–63 um) on the emergent model for ecotoxicology, the amphipod Parhyale hawaiensis, during 30 days of exposure. The following life-history traits were measured: (1) survival, (2) specific growth rate (SGR), (3) reproductive performance (precopulatory pairing behavior, fecundity, and time to release neonates), (4) molting frequency, (5) F1 newborn offspring survival and (6) MP bioaccumulation. No significant mortality or molt was seen in any of the treatments. MP caused a reduction in SGR, being more pronounced in females. The time for precopulatory pairing was 3-fold longer in amphipods exposed to MP. Fecundity decreased by 50 %, and the time to release juveniles was 6.7 days longer for amphipods exposed to MP. Finally, neonate survival decreased by 80 % after ten days of release. MP disrupts the reproductive mechanisms and triggers adverse effects on life history traits in P. hawaiensis.

Evaluation of precopulatory pairing behaviour and male fertility in a marine amphipod exposed to plastic additives

Plastics contain a mixture of chemical additives that can leach into the environment and potentially cause harmful effects on reproduction and the endocrine system. Two of these chemicals, N-butyl benzenesulfonamide (NBBS) and triphenyl phosphate (TPHP), are among the top 30 organic chemicals detected in surface and groundwater and are currently placed on international watchlist for evaluation. Although bans have been placed on legacy pollutants such as diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP), their persistence remains a concern. This study aimed to examine the impact of plastic additives, including NBBS, TPHP, DBP, and DEHP, on the reproductive behaviour and male fertility of the marine amphipod Echinogammarus marinus. Twenty precopulatory pairs of E. marinus were exposed to varying concentrations of the four test chemicals to assess their pairing behaviour. A high-throughput methodology was developed and optimised to record the contact time and re-pair time within 15 min and additional point observations for 96 h. The study found that low levels of NBBS, TPHP, and DEHP prolonged the contact and re-pairing time of amphipods and the proportion of pairs reduced drastically with re-pairing success ranging from 75% to 100% in the control group and 0%–85% in the exposed groups at 96 h. Sperm count declined by 40% and 60% in the 50 μg/l and 500 μg/l DBP groups, respectively, whereas TPHP resulted in significantly lower sperms in 50 μg/l exposed group. Animals exposed to NBBS and DEHP showed high interindividual variability in all exposed groups. Overall, this study provides evidence that plastic additives can disrupt the reproductive mechanisms and sperm counts of amphipods at environmentally relevant concentrations. Our research also demonstrated the usefulness of the precopulatory pairing mechanism as a sensitive endpoint in ecotoxicity assessments to proactively mitigate population-level effects in the aquatic environment.

Guarding success of the rarest sex in the amphipodHyalella aztecaSaussure, 1858 (Amphipoda: Hyalellidae)

AbstractTraditional methods to study precopulatory mate guarding in Hyalella azteca (Saussure, 1858) have some limitations because precopulatory pairs are usually observed in isolation. This condition precludes researchers from testing the role of the social environment in the duration of precopulatory mate guarding. We present the use of a sampling method to investigate the influence of sex ratios on guarding success (the proportion of precopulatory pairs per trial) in mixed-sex groups of H. azteca. The presence/absence of precopulatory pairs over eight consecutive days, morning and afternoon samplings, was analysed as a series of successes (runs) to estimate the mean precopula duration. The non-random nature of the observed duration of the precopula was tested against a probabilistic distribution based on the set of all ways every possible duration may occur. While skewed sex ratios increased guarding success of the rarest sex, even sex ratios decreased it. The mean duration of observed precopulas (2.32 and 2.08 d for the morning and afternoon periods, respectively) is consistent with theoretical and empirical results, which confirms the accuracy and usefulness of the sampling method we used. The use of this method and the associated probabilistic analysis of the relevant data can enhance the study of the effect of biotic and abiotic factors on precopulatory mate guarding. It can also be used to test predictions derived from hypothesis concerned with mate guarding among crustaceans.

Distribution patterns at different spatial scales reveal reproductive isolation and frequent syntopy among divergent lineages of an amphipod species complex in Western Carpathian streams

AbstractRecent discoveries of vast cryptic diversity among various aquatic invertebrates pose a challenge for understanding their ecology and biodiversity patterns, as well as for conservation. We studied the distribution, potential hybridization and habitat preferences of divergent lineages of the hyperdiverse Gammarus fossarum species complex (Crustacea: Amphipoda) in their contact zone in the Western Carpathians (Central Europe), to obtain insights into processes that may influence lineage coexistence. We initially screened over 60 sites to assess the regional distribution, confirming the presence of four lineages in streams of the study area. Two to three lineages were found to be syntopic at almost half the sites. Subsequently, nine such localities were studied in detail for the mesohabitat distribution (fast vs. slow water flow), pairing preferences, and temporal stability of the Gammarus community. A significant fraction of regional spatial variation was explained by the position in the dendritic river network, altitude, stream width, and anthropogenic stress. At the local scale, however, we did not find differences in distribution between mesohabitats with contrasting flow rates. Mixed precopulatory pairs in syntopy were extremely rare, even under highly imbalanced lineage ratios, confirming a strong prezygotic reproductive barrier between the studied lineages. This was congruent with species delimitation analyses using molecular markers, which confirmed the absence of any recent gene flow. The frequent and apparently temporally stable syntopy of these reproductively isolated Gammarus lineages warrants further research on the processes that may facilitate this coexistence, focusing on potential finer differences in their ecology, functional morphology, or biotic interactions.

Population and reproductive traits of a freshwater amphipod (Crustacea, Peracarida, Hyalellidae) from northwest of the state of Rio Grande do Sul, Brazil

Abstract The study of population and reproductive traits provides information about the ecological structure of natural populations. This study aimed to characterize dynamics and reproductive traits of Hyalella palmeirensis from a natural pond from southern Brazil. The amphipods were sampled monthly (August 2012 to July 2013) by a person with the aid of a hand net for 20 minutes. Ovigerous females and pre-copulatory pairs were individualized in the field. A total of 12,325 individuals were sampled, being 1,421 males, 6,983 females (including 215 ovigerous females) and 3,921 juveniles. Paired and unpaired males were significantly greater in size than females. There was a positive correlation between body size (CL) of paired males and females. Males and females showed bimodal distribution. Total sex ratio favored females, and these were more frequent in almost all months. Ovigerous females and precopulatory pairs were found throughout the year, but with high frequency in winter and autumn, respectively, characterizing a seasonal reproduction. Juveniles were sampled throughout the year, with greater intensity in the spring. The mean fecundity was 19.6 ± 4.34 eggs. No reduction in the number of eggs was observed during embryonic development. The results observed in H. palmeirensis demonstrate that this species has a population and reproductive dynamics very similar to other species of Hyalella already analyzed in southern Brazil. Moreover, it can be seen that although the H. palmeirensis occurs in an environment with anthropic influence (soy cultivation,) the population is managing to remain in the area, with reproduction and recruitment in most months of year.

Reproductive strategies of a population of a freshwater amphipod (Crustacea, Amhipoda, Hyalellidae) from southern Brazil

Abstract: The aim of this study was to characterize the reproductive strategies of a population of Hyalella bonariensis from southern Brazil. Pairing success, reproductive period of males and females, fecundity, and body size at the onset of reproduction were evaluated. Animals were sampled four times (August 2012, October 2012, January 2013, and April 2013) with the 250 µm-mesh dip net during 20 minutes by only one person. In the field, precopulatory pairs and ovigerous females were individualized. In the laboratory, cephalothorax length (CL) were measured and eggs and juveniles were removed from the females' marsupium and counted. The mean CL of paired males and females was significantly higher than that of non-paired males and females. A sexual dimorphism in body size was observed in the population - both paired and non-paired males were larger than females. Probably larger males have a higher probability of losing females during precopulatory behavior. A significant correlation was observed between the size of paired males and females - larger males often paired with larger females and smaller males paired with smaller females (r = 0.81). The pairing success of males increased with body size and we can assume that males from all size classes are able to find mates. The pairing success of females was independent of body size - paired and non-paired females had similar mean CL. The reproductive success increased with body size in males and females, and was more evident in males. Females from the largest size classes had null reproductive success. The idea that larger females can produce more eggs but might have a lower probability of finding a mate than smaller females was corroborated by our results. Therefore, smaller females have higher pairing success because they are capable of mating with a higher percentage of males. The estimated mean fecundity of H. bonariensis was 17.4 (± 3.89) eggs/juveniles. Our results are similar to those of other species of Hyalella from Brazil.

Reproductive biology of two species of Hyalella Smith, 1874 (Crustacea: Amphipoda: Hyalellidae) from southern Brazil

ABSTRACTReproduction is a critical event in the life cycle of animals, and a high amount of energy is allocated to it. The aim of this study was to characterize the reproductive biology of two recently described species of Hyalella from the north-east region of state of Rio Grande do Sul in southern Brazil. The specimens were collected during 12 months from a stream source (Hyalella georginae) and from a water reservoir (H. gauchensis), in Palmeira das Missões municipality, state of Rio Grande do Sul. Upon sampling, ovigerous females and pairs in precopulatory behaviour were separated into microtubes. In the laboratory, the cephalothorax length (CL in mm) of all specimens was measured. Male size at onset of sexual maturity was larger than that of the females, in both species. There was a correlation between the size of paired males and females: larger males carried larger females, in both Hyalella species. Precopulatory pairs were found throughout the year; however, H. georginae had a higher frequency in spring, and H. gauchensis in autumn. There was a positive correlation between the CL of ovigerous females and the number of eggs/juveniles. A reduction in the number of eggs throughout the embryonic development was observed in H. georginae. The average fecundity of H. georginae was significantly higher than H. gauchensis. The differences between the reproductive biology of these species may be related to the physical and environmental conditions at which they are exposed in their habitats.

Correlations between estrogen and testosterone concentrations, pairing status and acanthocephalan infection in an amphipod

AbstractMechanisms by which parasites induce phenotypic alterations are poorly understood in many host–parasite systems. Amphipods are an intermediate host for a range of acanthocephalan parasites, and there is substantial evidence that parasites manipulate the physiology and behavior of amphipods in a variety of ways, including significant reproductive impacts. Evidence suggests such manipulations may result from disruption of serotonergic and dopaminergic pathways. However, correlations with other hormones have not been widely investigated, in part because we lack understanding of the production and action of hormones such as estrogen and testosterone in amphipods. We investigated how estrogen (17β‐estradiol) and testosterone concentrations vary between unparasitized male and female amphipods Gammarus pseudolimnaeus that were and were not in precopulatory pairs. We also investigated whether infection with the acanthocephalan Corynosoma sp. is correlated with differences in estrogen or testosterone concentrations when compared to unparasitized conspecifics. Our results indicate that estrogen and testosterone differ significantly between male and female amphipods, whether paired or unpaired, suggesting they may have sex‐specific functions. The biological relevance of these hormones merits increased attention in light of the prevalence of endocrine‐disrupting compounds in aquatic systems. Further, we found that encysted acanthocephalan parasites are correlated with elevated concentrations of estrogen and depressed concentrations of testosterone in male, but not female amphipods. This correlation suggests the need for a broader investigation of the mechanisms underlying phenotypic alterations of amphipod behavior and physiology by acanthocephalan parasites.

Pairing behavior and reproduction in Hyalella azteca as sensitive endpoints for detecting long-term consequences of pesticide pulses

The aim of the present study was to examine acute and delayed effects of pulse exposure of the pyrethroid pesticide, permethrin, on precopulatory pairs of Hyalella azteca. Pairs of H. azteca were exposed to a single 1h pulse of different nominal concentrations of permethrin: 0, 0.3, 0.9 or 2.7μg/L. During exposure, pairing behavior was observed, and during a 56 day post-exposure period the treatments were monitored for pairing behavior, survival and reproductive output. All permethrin-exposed pairs separated within minutes during exposure and shortly thereafter became immobile; however they regained mobility after transfer to clean water. The time to re-form pairs was significantly longer in all tested concentrations compared to the control, although all surviving pairs re-formed within the 56 day test period. Nevertheless not all pairs exposed to 0.9 and 2.7μg/L reproduced. Furthermore the numbers of juveniles produced by pairs exposed to 0.9 and 2.7μg/L, but not 0.3μg/L, were lower throughout the entire post-exposure period compared to the control groups, and the total numbers of juveniles produced during 56 days were significantly lower in organisms exposed to 0.9 and 2.7μg/L, but not 0.3μg/L, compared to the control groups. The long-term effects of short-term exposure on reproductive behavior of pairs could potentially have consequences for the population dynamics of H. azteca. However, since individual-level responses can both overestimate and underestimate effects at the population level, appropriate population models are needed to reduce the uncertainty in extrapolating between these levels of biological organization.

Reproductive interference via interspecific pairing in an amphipod species complex

Many species experience reproductive interference defined as interspecific mating interactions that reduce the fitness of individuals in at least one of the species. We examined reproductive interference among three cryptic amphipod species that co-occur in the vegetated zone of lakes. Because these animals form precopulatory pairs and males commonly have indiscriminant mating behavior, we predicted that (1) there would be interspecific pairing, (2) interspecific pairing would be more common when conspecifics were rare, and (3) interspecific pairing would be more common in species combinations where males are larger than females. Using color phenotypes to distinguish species, a novel discovery reported here, we conducted experiments to evaluate patterns of interspecific pairing. In a no-choice experiment (i.e., amphipods had access to mates of only one species), we observed a low rate (5 %) of interspecific pairing and 80 % of these cases involved males that were larger than females. In a second experiment where individuals had access to conspecific mates at varying relative abundance, only 0.04 % of the pairs observed were between heterospecifics, suggesting that the interspecific pairing observed in the no-choice experiment was an artifact of not having access to conspecific mates. Our results suggest that at least one sex has sufficient species-recognition abilities to circumvent the formation of interspecific pairs; therefore, reproductive interference may be minimal in these species.

DIRECT AND INDIRECT FITNESS CONSEQUENCES OF FEMALE CHOICE IN A CRUSTACEAN

Understanding the evolution and maintenance of female mate choice requires information on both the benefits (the sum of direct and indirect benefits) and costs of selective mating. In this study, I assessed the fitness consequences of female mate choice in a freshwater crustacean. In Hyalella amphipods, males attempt to form precopulatory pairs with females. Large males, bearing large posterior gnathopods, tend to be over-represented in precopulatory pairs. I show that females receive both direct (reduced risk of predation while paired) and indirect (sexy sons) benefits from mating with these males. Furthermore, the behavioral mechanisms used to filter male phenotypes carry no detectable energetic cost for females. Thus, females that choose males with successful phenotypes are expected to have higher Darwinian fitness than females that mate at random. This study shows that direct and indirect selection act together to favor large male size, which explains the sexual size dimorphism and size-based mating biases observed in this species.

Pairing and reproductive success in two sympatric species of Hyalella (Crustacea, Amphipoda, Dogielinotidae) from southern Brazil

This study aimed at characterizing pairing and reproductive success in relation to male and female sizes of the sympatric freshwater gammarideans Hyalella pleoacuta and H. castroi from southern Brazil. These amphipods exhibit precopulatory mate guarding, in which a male will carry a potential mate beneath its ventral surface, guarding the female for several days until it molts and lays its eggs. The specimens were collected monthly with nets, from November 2003 to July 2004 in two trout aquaculture ponds at Sítio Vale das Trutas locality, São José dos Ausentes County, southern Brazil. The precopulatory pairs and ovigerous females were identified and separated in the field. In the laboratory, they were measured (cephalothorax length in mm), using a micrometer eyepiece in a stereoscopic microscope. Pairing success was estimated from the proportion of mating males and females related to their respective non-pairing individuals by size classes. Reproductive success was estimated from egg production. The mean cephalothorax length of paired males was larger than that of the unpaired males. For females, however, body size not affect pairing success for either species, because mean cephalothorax length of paired females did not differ significantly from unpaired females. Paired and unpaired males of both species of Hyalella were larger than the females. Positive assortative mating by size was observed in both species; i.e., larger males tended to pair with larger females. Male pairing success increased sharply with size. In both species, reproductive success in males increased with body size; however, the females of intermediate size classes showed greater reproductive success. This result supports the hypothesis that loading constraints play a part in structuring size-assortative pairing in these species.

Unnatural selection: Effects of a male-focused size-selective fishery on reproductive potential of a blue crab population

A male-focused size-selective fishery, like the one targeting the blue crab ( Callinectes sapidus Rathbun) along the Atlantic and Gulf coasts of the U.S., has the potential to reduce the average size of the males in the population, reduce the density of males in the population, and/or raise the ratio of females to males. All of these may affect the mating dynamics of the population by reducing the amount of sperm that males provide to females and decreasing the number of males available for copulation. We investigated the effect of the fishery on a blue crab population in upper Chesapeake Bay by collecting crabs in areas of markedly different fishing pressure. Crabs were taken as individuals and as mating pairs, which permitted assessing the size of males currently mating in nature, and the seminal resources they possess and transfer to females. Average size of males in subpopulations that have been subjected to heavier fishing pressure is indeed smaller, and the smaller males pass less sperm and accessory fluid to females than would larger males if they were still present. Some males in pre-copulatory pairs are as sperm depleted as males that had just completed copulation, indicating that they are mating more frequently than they can replace their seminal resources. The most sperm-depleted males in the population are not even pairing or attempting to mate.

Does social isolation during early development affect the reproductive behaviour of the amphipod Hyalella azteca (Saussure, 1858)?

Hyalella azteca, in common with other amphipods, exhibits precopulatory pairing, when the male carries the female beneath his ventral surface for several days until she moults, and so ensures successful sexual contact during the post ecdysis period when mating can occur. Although animal courtship is a ritualist behaviour, for some groups there is clear evidence that mating behaviour and mate selection are dependant upon and can be influenced by experiences and learning during early development stages. This study was designed to examine the effect of social isolation, during neonatal development, on the success of H. azteca in forming precopula pairs for the first time. The results demonstrated that animals reared in isolation and so deprived of physical, visual and chemical exposure to conspecifics formed precopula pairs at the same developmental time and with the same frequency as animals which had been reared together with their conspecifics.

Reduced Recruitment in Hyalella azteca (Saussure, 1858) Exposed to Copper

Neonates of the amphipod Hyalella azteca were exposed for a 35-day period in the laboratory to a range of copper concentrations, nominally 18μg/l, 40μg/l, 70μg/l and 260μg/l. The reproductive status of the population was assessed by recording recruitment, the number of precopulatory pairs and number of gravid females. At the end of the experiment, the body lengths of individuals were measured using image analysis. There was a significant decrease in the final population size of H. azteca with increasing copper concentration and compared with the control. Copper significantly reduced recruitment of juveniles and length composition of the final population and there was also a trend toward reduced precopula number with increasing copper concentrations.

MATURITY IN MALE STONE CRAB HAPALOGASTER DENTATA (ANOMURA: LITHODIDAE) AND ITS APPLICATION FOR FISHERY MANAGEMENT

Abstract Male size at maturity is important information for management of crab fisheries. Morphometric and/or gonadal maturity, which have been used to estimate size at maturity for many male crustaceans, were examined as potential indicators of functional maturity using small, unfished stone crab Hapalogaster dentata as a model species. Morphometric size of maturity as estimated by the change of the allometric relationship between carapace length (CL) and chela height was 5.2 mm CL. The size at gonadal maturity as estimated by histological examination of the gonad and vasa deferentia was 5.3 mm CL. The functional size at maturity as estimated by observing precopulatory guarding pairs in the field was 9.5 mm CL, much larger than the former two estimates. Size-assortative mating was observed between guarding males and guarded females. Female choice for larger males and advantage of the larger males in mating fights may be the reasons why such discrepancy between morphometric and/or gonadal maturity and fun...

Concurrent Signals and Behavioral Plasticity in Blue Crab (Callinectes sapidus Rathbun) Courtship.

Behavioral flexibility and behavioral regulation through courtship signals may both contribute to mating success. Blue crabs (Callinectes sapidus) form precopulatory pairs after courtship periods that are influenced by female and perhaps male urine-based chemical signals. In this study, male and female crabs were observed in 1.5-m circular outdoor pools for 45 min while the occurrence and sequence of courtship behaviors and pairing outcomes were recorded. These results were then compared with trials in which males or females were blindfolded; lateral antennule (outer flagellum) ablated; blindfolded and lateral antennule ablated; or had received nephropore blocks. The relative importance of visual and chemical sensory systems during blue crab courtship were then determined and urine and non-urine based chemical signals for both males and females were examined. Courtship behaviors varied considerably in occurrence and sequence; no measured behavior was necessary for pairing success. Male or female blindfolding had no effect on any measured behavior. Males and females required chemical information for normal courtship behaviors, yet blocking male or female urine release did not affect courtship behaviors. Males required chemical information to initiate pairing or to maintain stable pairs. Male urine release was necessary for stable pairing, suggesting that male urine signals may be involved in pair maintenance rather than pair formation. Females that could not receive chemical information paired faster and elicited fewer male agonistic behaviors. The results demonstrate a great variability and flexibility in blue crab courtship, with no evidence for stereotyped behavioral sequences. However, these behaviors appear regulated by urine- and nonurine-based redundant chemical signals emanating from both males and females. Although urine-based signals play roles in blue crab courtship, chemical signals from other sites appear to carry sufficient information to elicit a full range of behavioral responses in males and females.

The acute toxicity of lindane to hyalella azteca and the development of a sublethal bioassay based on precopulatory guarding behavior

Acute and sublethal toxicity of the organochlorine insecticide lindane to the amphipod crustacean Hyalella azteca was investigated. Acute experiments were conducted for a maximum test exposure period of 240 h with adult and neonate H. azteca. Median lethal concentrations (LC50s) determined for adult Hyalella included a 48-h LC50 of 47.6 &mgr;g/L and 240-h LC50 of 26.9 &mgr;g/L. For neonate H. azteca 24-, 48-, and 240-h LC50s were 29.5, 14.8, and 9.8 &mgr;g lindane/L, respectively. Neonate H. azteca were approximately three times more sensitive than adults. Two sublethal toxicity bioassays were developed based on the direct and indirect disruption of the precopulatory or mate guarding behavior of Hyalella. This reproductive behavior is readily quantifiable and of ecological significance as it is a vital component of the mating success of the species. The direct disruption bioassay examined the separation of precopulatory pairs maintained in control water and a range of lindane concentrations during a 24-h exposure period. Median separation times (ST50s) were determined and the LOEC was 24.4 &mgr;g lindane/L. The indirect disruption bioassay consisted of a test exposure period of just 4 h after which an invertebrate anesthetic solution was administered to induce separation of precopulatory pairs. The LOEC was 17.3 &mgr;g lindane/L, suggesting that the indirect precopulatory separation bioassay was comparable to the 24-h direct separation study. Both bioassays are rapid, relatively simple to perform, and have yielded effect concentrations that correspond with LC50 values determined using adult and neonate H. azteca life stages over more prolonged lindane exposures. Following some modification, these behavioral bioassays may be suitable for use in the hazard evaluation of sediments and for deployment as in situ toxicity tests.

The Influence of Chemical Stimuli from Predators on Precopulatory Pairing by the Amphipod, Gammarus pseudolimnaeus

AbstractMany amphipod crustaceans exhibit precopulatory mate guarding. Field samples of the amphipod Gammarus pseudolimnaeus indicated that pairs were positively size assortative. Receptive individuals readily formed pairs in the laboratory and the latency to formation of precopulatory pairs was decreased under threat of predation. In addition, females and, under conditions of extreme danger, males that formed pairs were significantly smaller when under the threat of predation. Amphipods distinguished between chemical stimuli (aquarium water) from predatory and nonpredatory fishes and between chemical cues from fish predators (trout) that had recently eaten conspecific amphipods vs. those fed a control diet of pelleted commercial fish food. These data indicate that chemical stimuli associated with predators can influence reproductive behavior of amphipods. The results also suggest the hypotheses that: 1. search time may be costly in terms of probability of predation; and 2. small pairs may be safer from predation than larger pairs.

Disruption of precopula in Gammarus pulex as a result of brief exposure to Gamma-hexachlorocyclohexane (Lindane)

Precopulatory pairs of Gammarus pulex were exposed to each of 3 concentrations (0.5; 1.0; 2.0 mg 1 −1) of Gamma-hexachlorocyclohexane (Lindane) for each of 3 exposure periods (2; 10; 20 min). A total of 14 treatments (including 5 controls) was tested each of them involving 24 individual pairs. Premature disruption of precopula as a direct result of exposure occurred in the exposure treatments 0.5; 1.0; 2.0 mg 1 −1 (20 min), 1.0 and 2.0 mg 1 −1 (10 min), and 2.0 mg 1 −1 (2 min). The disruptive effect was more apparent during the first hours of post-exposure, and at 2 h, disruption of precopula ranged from 60.0 – 95.8%. Recovery of precopula occurred to different extents in all affected treatments from 4 to 72 h after exposure. The median precopula duration, LP 50 (h) was estimated for 11 of 14 treatments, and also of recently formed pairs in contaminant-free artificial pond water at 15° C.