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Rivers suffer from multiple stressors acting simultaneously on their biota, but the consequences are poorly quantified at the global scale. We evaluated the biological condition of rivers globally, including the largest proportion of countries from the Global South published to date. We gathered macroinvertebrate- and fish-based assessments from 72,275 and 37,676 sites, respectively, from 64 study regions across six continents and 45 nations. Because assessments were based on differing methods, different systems were consolidated into a 3-class system: Good, Impaired, or Severely Impaired, following common guidelines. The proportion of sites in each class by study area was calculated and each region was assigned a Köppen-Geiger climate type, Human Footprint score (addressing landscape alterations), Human Development Index (HDI) score (addressing social welfare), % rivers with good ambient water quality, % protected freshwater key biodiversity areas; and % of forest area net change rate. We found that 50% of macroinvertebrate sites and 42% of fish sites were in Good condition, whereas 21% and 29% were Severely Impaired, respectively. The poorest biological conditions occurred in Arid and Equatorial climates and the best conditions occurred in Snow climates. Severely Impaired conditions were associated (Pearson correlation coefficient) with higher HDI scores, poorer physico-chemical water quality, and lower proportions of protected freshwater areas. Good biological conditions were associated with good water quality and increased forested areas. It is essential to implement statutory bioassessment programs in Asian, African, and South American countries, and continue them in Oceania, Europe, and North America. There is a need to invest in assessments based on fish, as there is less information globally and fish were strong indicators of degradation. Our study highlights a need to increase the extent and number of protected river catchments, preserve and restore natural forested areas in the catchments, treat wastewater discharges, and improve river connectivity.

The intermediate stages of speciation are important for understanding the processes involved in the creation of biodiversity, and also comprise a number of interesting phenomena. However, difficulties are associated with dividing clear speciation stages because speciation is a continuous process. Therefore, the elucidation of speciation is an interesting and important task in evolutionary biology. We herein present an example of a species in an intermediate stage of speciation using the giant water bug Appasus japonicus (Heteroptera, Belostomatidae) that was investigated using mating experiments and phylogenetic analyses of the mtDNA COI (658 bp) and 16S rRNA (435 bp) regions, and nDNA SSR (13 loci) and its genome-wide SNPs (11,241 SNPs). The results of our phylogenetic analyses based on their mtDNA dataset and the genome-wide SNPs dataset strongly supported the paraphyly of the Japanese populations. Therefore, it is suggested that their ancestral lineage which being distributed in the Japanese Archipelago subsequently migrated to the Eurasian Continent (i.e., "back-dispersal" occurred). Furthermore, the results of the mating experiments suggested that among A. japonicus, even between closely related lineages, pre-mating reproductive isolation has been established by the differentiation of copulatory organ morphologies. In contrast, pre-mating reproductive isolation is not established in the absence of the differentiation of copulatory organ morphologies, even if genetic differentiation is prominent. These results suggested that their phylogenetic distance does not predict pre-mating reproductive isolation. Furthermore, in the present study, we present a clear example of pre-mating reproductive isolation driving speciation between closely related lineages.

Chironomid larvae are among the dominant benthic macroinvertebrates in all types of water systems in South Korea. They may pass through pipes in rivers (raw water) and occur in drinking water, thus creating public health issues. However, little is known about the larval stages of chironomids in large South Korean rivers. Therefore, we examined larval–adult associations in chironomids inhabiting major rivers used as water sources. The larvae were collected in 2015 and 2016 from nine locations along the four largest rivers in South Korea using a Ponar grab. Cytochrome oxidase subunit I (COI) sequences were generated from the larval specimens, and the species were identified by comparing these sequences to those in a newly constructed DNA barcode library of Chironomidae in South Korea. The samples from the four rivers yielded 61 mitochondrial COI sequences belonging to 18 species, including Hydrobaenus kondoi Saether, 1989, which was reported for the first time in the Korean Peninsula. Further, morphological identification of the larvae was conducted, and a pictorial taxonomic key to Chironomidae species in large rivers in South Korea was developed to facilitate freshwater biomonitoring research. Finally, an action flow chart was created for the rapid identification of chironomid larvae in infested drinking water or water purification facilities.

Since its introduction to Asia

Studying the phenotypic variation of organisms along environmental gradients can provide insight into the influences of specific environmental factors. Mongolia, which is distributed across three different biomes, is an ideal location for studying the mechanisms that underlie such phenotypic variation over a large range of climate. The present study examined the variation in shape and size of the body in a ball-rolling dung beetle, Gymnopleurus mopsus (Pallas), in Mongolia and investigated the effects of climate on the species’ morphology. A total of 290 individuals were collected from seven sites and were analysed using multivariate and regression approaches, as well as geometric morphometrics. Body shape and size varied across the study sites and between the different biomes. Populations from the desert-steppe region had thinner bodies and longer heads than those from the steppe region, possibly to facilitate burrowing. Variation in the species’ body size followed a pattern that was the converse of Bergmann’s rule and, thus, might increase heat capacity and enhance thermoregulation ability in the desert-steppe region. Accordingly, the results of the present study provide novel insight into the influence of climate on the variation of dung beetle phenotypes.

The water penny beetles (Coleoptera: Psephenidae) of the Korean Peninsula are reviewed based on their external morphology and mitochondrial cytochrome c oxidase subunit I gene sequences. Four subfamilies, four genera, and four species, including a new species described herein, are recognized: Eubrianax ramicornis Kiesenwetter, 1874 (Eubrianacinae), Homoeogenus coreanicum sp. nov. (Eubriinae), Malacopsephenoides japonicus (Masuda, 1935) (Psephenoidinae), and Mataeopsephus japonicus (Matsumura, 1916) (Psepheninae). The psephenid subfamily Eubriinae and the genus Homoeogenus Waterhouse, 1880 are here recorded from the Korean Peninsula for the first time. Analyses of the cytochrome c oxidase subunit I gene sequences of larvae and adults of the four Korean species produced a bootstrap support of 100% for each species. Descriptions, habitat data, habitus photographs, line-drawings of male genitalia, and keys to the adults and larvae of the Korean species of Psephenidae are provided. Keywords: DNA barcode, Homoeogenus coreanicum sp. nov., Korean Peninsula, Taxonomic review, Water penny beetles

Due to the increasing mosquito activity and the risks associated with them, mosquito control has become a global issue. In particular, biological control using the natural enemies of mosquitoes has attracted great attention. Hydrochara affinis, an aquatic beetle, has recently been identified as a biological control agent for mosquitoes. It is necessary to explore the environmental characteristics of its natural habitats for successful mosquito control. Therefore, we surveyed the environmental characteristics of its natural habitats (dwelling sites, DS), former habitats (former dwelling sites, FDS), and non-habitats located near its natural habitats or with a similar landscape (non-dwelling sites, NDS). Perennial emergent macrophytes accounted for 85.5% of the total plant cover in the DS, while they accounted for 44.2% and 43.0% in FDS and NDS, respectively. The environmental characteristics, such as electrical conductivity and cation concentration of the water, in the 3 categories (DS, FDS, and NDS) were significantly different. The ranges of these values in the DS were wide. Based on these results, we concluded that perennial emergent macrophyte communities can be a determinant for H. affinis habitat selection. It is, therefore, important to maintain a proper environment in which macrophyte communities, that can serve as habitats for H. affinis, can proliferate.

Predators and their interactions with target prey influence the efficiency of control strategies. In the present study, we demonstrate the implementation of natural predator selection for controlling dengue vectors in northern Vietnam through field-based observation of aquatic insect predators in natural habitats and lab-based assessment of predatorial capacities for several aquatic insect predators. The selected species was then used to evaluate the predatory-prey interaction using functional responses (FRs) toward 3rd- and 4th-instar larvae of four major medical mosquito species (Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus). The preference of selected predators for Ae. aegypti larvae over other mosquito larvae was also investigated. Both field observation and lab experiments indicated that the giant water bug Diplonychus rusticus was abundant and exhibited the highest predatory capacity for mosquito larvae. The predator exhibited type II FRs when offered each of the four prey species, and the greatest attack rates were observed for Ae. aegypti and Ae. albopictus, with only negligible differences observed in the handling times of the prey species. Further, Manly's selectivity (α) values calculated from the prey choice experiments showed that Ae. aegypti was preferred over both Cx. quinquefasciatus and An. minimus. Together, these findings indicate that D. rusticus could be successfully used to facilitate the biological control of both Ae. aegypti and Ae. albopictus within the species' distributional overlap in Southeast Asia.

Aedes albopictus is a native mosquito to Southeast Asia with a high potential for disease transmission. Understanding how Ae. albopictus populations that develop in the species’ native range is useful for planning future control strategies and for identifying the sources of invasive ranges. The present study aims to investigate the genetic diversity and population structure of Ae. albopictus across various climatic regions of Vietnam. We analyzed mitochondrial cytochrome oxidase I (COI) gene sequences from specimens collected from 16 localities, and we used distance-based redundancy analysis to evaluate the amount of variation in the genetic distance that could be explained by both geographic distance and climatic factors. High levels of genetic polymorphism were detected, and the haplotypes were similar to those sequences from both temperate and tropical regions worldwide. Of note, these haplotype groups were geographically distributed, resulting in a distinct population structure in which northeastern populations and the remaining populations were genetically differentiated. Notably, genetic variation among the Ae. albopictus populations was driven primarily by climatic factors (64.55%) and to a lesser extent was also influenced by geographic distance (33.73%). These findings fill important gaps in the current understanding of the population genetics of Ae. albopictus in Vietnam, especially with respect to providing data to track the origin of the invaded regions worldwide.

Recent molecular analyses of transcriptome data from 94 species across 92 genera of North American Plecoptera identified the genus Kathroperla Banks, 1920 as sister group to Chloroperlidae + Perlodidae. Given that the genus Kathroperla has historically been included as a member of the family Chloroperlidae, this discovery indicated further investigation of the genus and the subfamily Paraperlinae was needed. Both transcriptome and genome sequencing datasets were generated from 32 species of the infraorder Systellognatha, including all described species of the Paraperlinae, to test the phylogenetic placement of these taxa. From these datasets, a large phylogenomic data matrix of 800 orthologous genes was produced, and multiple analyses were conducted, including both concatenated and coalescent analyses. Morphological comparisons were made among all Paraperlinae using light microscopy. All molecular results support a monophyletic Kathroperla, which is supported as sister taxon to the remaining Perloidea by five of six molecular analyses. Postocular head length is determined to be a distinct morphological character of this genus. Combined molecular and morphological evidence support the designation of Kathroperlidae, fam. n., as the seventeenth family of extant Plecoptera.

The genus Ephoron Williamson, 1802 is widely distributed around the world. In Thailand, only Ephoron indicus Pictet, 1843 was reported in 1961. In this study, a new Ephoron mayfly is described as Ephoron ookaewae sp. nov. In this new species description, the morphological characteristics of larvae and eggs in addition to adults are also shown in detail. Number of tubercles on mandibular tusks, and a median frontal process in larvae, distinguish Ephoron ookaewae sp. nov. from other Ephoron species. Their polar cap shape and a concave indentation in their eggs are also unusual. In addition, the mitochondrial DNA COI sequence data of the newly described Ephoron ookaewae sp. nov. is registered in GenBank. Registration of sequence data for the DNA barcode region of Ephoron mayflies inhabiting the Oriental region remains limited, however it will be useful for future research.

Simple Summary The critically endangered Callipogon relictus is one of the largest longhorn beetle species and also the only remnant species of the genus Callipogon found in the Palearctic region. Knowledge of the phylogeographic history and current conservational status is essential to understand its unique distribution and to establish efficient conservation strategies. We studied the phylogeographical pattern and genetic diversity of C. relictus from almost its entire geographical range. The level of genetic diversity and divergence of C. relictus from the known distributions in four regions in Russia, China, North Korea, and South Korea using two mitochondrial markers (COI and COII) revealed that genetic diversity lies within the range of intraspecific levels. Relatively high genetic diversity with no distinct phylogeographic structures indicates that the wide range of current fragmented populations might be the remnant of genetically diverse populations in the past. The results of our phylogenetic and population genetic analyses suggest that C. relictus populations surveyed in the present study can be used as a “source” for a future restoration and conservation program. The findings of the genetic characteristics and phylogeographic history of C. relictus will help to establish effective conservation strategies for this endangered longhorn beetle species in Northeast Asia. Abstract The longhorn beetle, Callipogon (Eoxenus) relictus Semenov, is the only remnant species found in the Palearctic region, while all other Callipogon species are distributed mainly in Central America and partly in South America. This species has been placed in the first category (as ‘critically endangered’) of the Red Data Book in Russia and designated as one of the top-priority target species among all endangered invertebrate species for restoration in South Korea since 2006. Although its restricted distribution in Northeast Asia with a high conservational value has been highlighted, genetic features of C. relictus from different geographic regions remain unexplored. We first investigated the level of genetic diversity and phylogeographic patterns of C. relictus to evaluate the current conservational status and the feasibility of the implementation of a restoration program. The average genetic divergence of mitochondrial gene COI based on Kimura-2-parameter distance among the four regions in Russia, China, North Korea, and South Korea was 2.2%, which lies within the range of intraspecific levels. However, two separate clades with 3.8% divergence were identified, despite no geographical clustering of haplotypes. The linear pattern of the haplotype network with a high level of haplotype and nucleotide diversities suggests that the wide range of currently fragmented populations might be the remnant of genetically diverse populations in the past. This study will provide crucial information on the genetic characteristics and phylogeographic history of C. relictus, which will help to establish conservation strategies for this cherished insect species in Northeast Asia.

Ips subelongatus is a species of bark beetle experiencing population outbreaks in Korea. In this study, a predictive model and empirical prediction were used to forecast the spring flight of these beetles in Japanese larch forests. The number of beetles caught in pheromone traps was investigated in larch forests thinned in 2009, 2010, 2012, or 2013. Data from the sites thinned in 2009, 2010, or 2012 were used in the predictive model based on a degree-day model that was validated using data from the site thinned in 2013. The lower threshold temperature for flight (LTF) and a thermal sum for the spring flight of I. subelongatus were estimated. The empirical prediction that beetles initiate their flight when daily maximum temperatures reach 16 or 20°C was tested using daily maximum temperature and the beetles caught. The LTF was estimated as 5.97°C, with 42.95 degree-days required for initiation of spring flight. The median flight dates were estimated with a discrepancy from 1 to 3 d by the predictive model. Using the empirical prediction, differences between the day when daily maximum temperature reached 16 or 20°C and flight peak days ranged from 4 to 45 d. These results demonstrate that the predictive model is more suitable than the empirical prediction for predicting the spring flight of I. subelongatus. Overwintering I. subelongatus adults seem to need to gain a determined thermal sum before initiating spring flight rather than merely waiting for the daily maximum temperature to exceed a critical temperature.

The phylogenetic relationships among the members of Macronematinae (Trichoptera: Hydropsychidae) have long been debated and often revised. Our study based on a large subunit (28S) nuclear ribosomal DNA (D2 expansion fragment; 464 base pairs) of gene sequences found all genera to be monophyletic except for the genusPolymorphanisusWalker, which produced two clades in accordance with its species groups established using morphological characters. Phylogenetic reconstruction based on mitochondrial cytochrome oxidase I (658 base pairs) found the same monophyletic relationships, however, except for the generaPolymorphanisusandMacrostemumKolenati. The placement of the genusLeptonemaGuérin-Méneville as the basal clade in the subfamily was also ruled out, which is consistent with previous morphological findings. The monophyly of two previously recognised tribes (Macronematini and Polymorphanisini) was not supported. We also found that the generaCentromacronemaUlmer,BaliomorphaNeboiss, andMacronemaPictet are most closely related, having three head setal warts on the vertex of the head and unsegmented inferior appendages of male genitalia as their synapomorphies. Our study supported the hypothesis of close relationship of the generaAmphipsycheMcLachlan andProtomacronemaUlmer with the morphological synapomorphies: absence of a forewing discoidal cell and the presence of elevated head carinae on the larvae. Some notable evolutionary novelties in the structure of adults in various lineages of this subfamily include reduction of maxillae and labium, reduction and loss of a discoidal cell and evolution of ac-sccrossvein in each forewing, fusion of segments in each male inferior appendage, and evolution of dark colour patterns in the forewings. Also, the head setal warts, which are unique organs in Trichoptera, evolved towards a decreasing number in the subfamily. This first molecular phylogenetic study, covering most genera in Macronematinae and including an interpretation of 12 key evolutionary novelties, provides an important basis for resolving long-standing questions regarding phylogenetic relationships and classification of genera and species and helps lay a stronger foundation for inference of testable hypotheses about functional traits for species of this common and widespread subfamily.

The Northeast Asian species of Kisaura Ross, 1956 (Philopotamidae) are re-examined and males and females of a new species from South Korea are described and illustrated. Kisaura coreana sp. nov. closely resembles K. tsudai (Botosǎneanu, 1970) and K. hattorii (Kuhara, 1999) described from Japan, but distinguished from the latter two species by the shape of the spiniform processes of segment X in the male genitalia. Further, we conclude that K. kisoensis (Tsuda, 1939) is not a synonym of K. aurascens (Martynov, 1934) because of differences in the genitalia, which was also supported by molecular data. Also, the Neighbor Joining tree shows a clade including both the Japanese species K. nozakii (Kuhara, 1999) and K. borealis (Kuhara, 1999) with high bootstrap values, especially that for K. nozakii, collected from Hoshu Shiga, Kanzaki-gawa, Kazakoshi-dani (99% bootstrap value). This high value could reflect the possibility that they are the same species and just having differences based on geographic variation in morphological traits. These two species will be the subject of future studies.

Callipogon relictus Semenov (Coleoptera: Cerambycidae) is currently listed in the Red Data Books (Category I) of Russia and South Korea and, in 2006, was designated by the South Korean government as the first invertebrate priority target species in a restoration project. However, the species is also classified as an invasive quarantine pest by the Canadian Food Inspection Agency. Due to the five-year to seven-year life cycle of the species, experimental information about instar numbers has been poorly documented. Therefore, the goal of the present study was to document the instar numbers of non-diapause Callipogon relictus larvae reared on an artificial diet. Under conditions of 30 °C, 60% relative humidity, and constant dark (0:24 hour light-dark photoperiod), developmental pathways of 8, 10, and 12 instars were observed. The effect of temperature (20, 25, and 30 °C) on the duration of larval development was also examined to identify the optimum temperature for producing Callipogon relictus for conservational purposes. Larvae reared at 30 °C and 60% relative humidity, without a chill period, developed in seven to eight months, which is about one-tenth the duration of C. relictus development under natural conditions and the most rapid development of C. relictus observed to date.

Mitochondrial genomes provide an additional data source for nuclear phylogenomics. Recently, the taxonomic classification of Ophiuroidea was changed dramatically based on a molecular phylog...

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing baseline reference for phylogenetic analysis and aquatic ecosystem biomonitoring. We obtained 112 novel sequences of the barcode region of the mitochondrial DNA cytochrome c oxidase subunit I gene representing 11 families, 25 genera, and 43 species of mayfly (Insecta: Ephemeroptera) from South Korea. No species shared barcode sequences and all can be identified with barcodes with a possible exception of some species. Minimum levels of interspecific genetic distances ranged from 6.7 to 32.9% (mean: 23.7%), whereas average levels of intraspecific divergence was 3.7%. The latter value was inflated by the presence of very high divergences within some taxa. In fact, approximately 33.3% (15/45) of the species included two or more haplotype clusters showing greater than 5.0% sequence divergence and some values were as high as 32.9%. Many of the species with high intraspecific divergences are para‐ or polyphyletic and represent the possibility of species complexes. Our study suggests that type or topotype specimens should be sequenced to identify accurate barcoding clusters with morphological species concepts and also to determine the status of currently synonymized species.

Korean oak wilt (KOW) is vectored by the beetle Platypus koryoensis (Murayama) (Coleoptera: Curculionidae), a native species of Korea, whose dispersal distance is a key factor determining the spread of damage by KOW. To estimate dispersal distance at stand level, we conducted a mark-release-recapture (MRR) experiment and validated its results using an independent data. Sticky traps were attached to the trunks of oak trees up to 48.8 m from the release point. Beetles were marked with different three fluorescent powders by date and released, and the number of recaptured beetles was counted 90 min after release. To validate the flight distance, annual mean dispersal distance of P. koryoensis population was analyzed using GPS coordinates of oak trees with the symptom of KOW recorded in the field from 2012 to 2014 in independent oak stands that have been damaged by KOW since 2012. The beetles were recaptured only on the day they were released, suggesting that the beetles only make one flight. The percentage of recaptured beetles was 6.0 ± 1.6%. The mean dispersal distance was 18.0 ± 1.3 m, and more than 85% of recaptured beetles were caught within 25 m. Annual movement distances in infested stands were 24.1 and 19.9 m from 2012 to 2013 and 2013 to 2014, respectively, similar to the dispersal distance obtained from our MRR experiment. Our results showed that the dispersal distance of P. koryoensis estimated by MRR is a useful process for predicting the spread of areas damaged by KOW.

The type locality (Korea) of the dung beetle Paragymnopleurus singularis (Waterhouse, 1890) (Gymnopleurini, Scarabaeidae, Coleoptera) has been questioned. For example, Genier (2003) synonymized P. singularis with P. maurus (Sharp, 1875) and considered P. maurus to be endemic to Borneo. Accordingly, the aim of the present study was to investigate the occurrence of P. singularis in Korea and to infer its actual type locality by reviewing available literature. Several faunistic studies have documented P. singularis in Korea, albeit without specimen confirmation. However, the species has not been collected in Korea since its original description. The voyage log of E. Belcher, who captained the H.M.S. Samarang from 1843 to 1846, suggests that the P. singularis type specimens were collected from Sarawak, Borneo, and mislabeled as specimens from Korea. Therefore, the actual type locality of P. singularis is likely Sarawak in Borneo, and Gymnopleurus mopsus (Pallas, 1781) is the only member of the tribe Gymnopleurini that actually occurs in Korea.

Hydrochara affinis (Coleoptera: Hydrophilidae), a water scavenger beetle, was recently identified as a natural and effective agent for biological mosquito control, it was reported to exhibit high rates of mosquito larvae predation. However, maintaining the quality (i.e., natural ecological attributes, such as genetic variation) of laboratory-reared populations is essential for ensuring the long-term success of biological control programs. Accordingly, here, we aimed to use mitochondrial Cytochrome c oxidase subunit I (COI) sequences to document the genetic diversity, population structure, and phylogenetic position of natural and lab-reared H. affinis populations in South Korea and use geometric morphometric analysis to investigate the populations&rsquo, morphological divergence. The natural H. affinis populations possessed high genetic diversity and numerous COI haplotypes, suggesting that these populations were healthy and could be directly applied to mosquito habitats without alterations to their natural genetic attributes. The lab-reared populations also possessed high genetic diversity and, thus, the potential for high adaptive capacity to new environments. Although no distinct population genetic structures were observed, quantitative variation was observed in the body shape of both the natural and lab-reared populations. The high levels of genetic and morphologic variation observed in the H. affinis populations examined here indicate the species&rsquo, favorable conservation status, genetic diversity, adaptive capacity, and, thus, &ldquo, suitability&rdquo, for field application as an effective mosquito control agent.

Populations of Gymnopleurus mopsus (family Scarabaeidae), a dung beetle that displays dung-rolling behavior (i.e., a telecoprid), have recently experienced sharp declines, and many populations are now at high risk of local extinction. However, Mongolia, which constitutes a major portion of the species’ distribution, still sustains a relatively large population. Here, we used mitochondrial COI sequences to investigate the within-population genetic diversity and both the genetic and phylogeographic structures of 24 G. mopsus populations across the species’ main distribution in Mongolia. Several lines of evidence indicated that the phylogeographic structure of G. mopsus had been influenced by a recent and sudden demographic expansion. Interestingly, the expansion of Mongolia’s G. mopsus population corresponded to the advent of livestock domestication in the region, and the species’ genetic structure coincided with road networks, which presumably serve as migration routes for livestock that might mediate the beetle’s dispersal. In addition, we also found that G. mopsus possesses high levels of haplotype diversity, which is generally indicative of large effective population sizes (Ne). Overall, the present study contributes to the current understanding of G. mopsus’ demographic history and dispersal patterns and also provides valuable information for the species’ conservation and management.

A new behavioral endpoint in ecotoxicity testing, burrowing behavior of Chironomus yoshimatsui larvae that survived pre-exposure to 0, 1, 5, and 10 mg/L of cadmium for 96 h was evaluated in a system composed of clean reconstituted water and sand for 5 h, at 30 min intervals. The effective time required for 50% of the introduced larvae to burrow (ET50), the proportion of the maximum burrowing rate reached during a 5 h period (BR5h), and the area under the curve at 5 h (AUC5h) were calculated based on the fitted relationship between burrowing rate and elapsed time. These effect parameters were chosen because they take into account either the burrowing speed of the larvae (ET50), or the proportion of the maximum burrowing rate reached during a 5 h period (BR5h), or both (AUC5h). We found that higher cadmium concentrations resulted in longer ET50 and lower AUC5h values. Additionally, field water samples were collected from streams near residential, natural, agricultural, and industrial areas in Ansan City, Korea in 2014 and 2015, and were used to validate the proposed burrowing test method. The results of the burrowing tests with field collected water samples also confirmed the sensitivity of burrowing activity as an ecotoxicity endpoint, indicated by the large differences among the ET50, BR5h, and AUC5h across the sampling sites and dates with industrial sites strongly affecting burrowing behavior. This study clearly showed that the proposed burrowing test could provide information that would not be detected with the existing acute toxicity tests, within a relatively short time, and can therefore be used as a complement to existing chemical analyses and acute toxicity tests.