Population dynamics in Daphnia Magna as modified by chronic tetradifon stress

Abstract

Two Daphnia magna offsprings (animals from the first and third brood) whose parentals (F₀‐generation) were exposed during 21 days to different tetradifon (4‐chlorophenyl 2,4,5‐trichlophenyl sulfone) concentrations, were transferred to a pesticide free medium during 21 days (recovery period). The algae Nannochloris oculata (5x10⁵ cells/mL) was used as food. In this recovery study, survival, growth and reproduction (mean total young per female, mean brood size, onset of reproduction and mean number broods per female) were assessed as individual parameters and the intrinsic rate of natural increase (r) as population parameter, for F₁ generation (1st and 3rd broods). Reproduction was still reduced in F₁ (1st and 3rd broods) generation daphnids from parentals (F₀) exposed to 0.18 mg/L tetradifon and higher concentrations even after 21 days in clean water. However, survival was not significantly different (p>0.05) in those F1 offsprings from parentals pre‐exposed to the acaricide. Growth was still reduced in F1 daphnids from parentals pre‐exposed to 0.10–0.44 mg/L tetradifon. The intrinsic rate of natural increase (r) was still affected in Fl generation daphnids, specially in those from the third brood. Therefore, Fl generation D. magna offsprings from a parental generation (F0) previously exposed to the acaricide tetradifon were not able to recuperate completely when a recovery period of 21 days was allowed.     

Acute, chronic and sublethal effects of the herbicide propanil on Daphnia magna

Acute and chronic toxicity tests with propanil were conducted on Daphnia magna. The 24 and 48 h LC50 were 43.74 and 5.01 mg/l respectively. Chronic toxicity tests were carried out using sublethal propanil concentrations (0.07, 0.10, 0.21 and 0.55 mg/l) during 21 days. The effect of propanil on survival, reproduction and growth of D. magna organisms was monitored. The parameters used to evaluate herbicide effect on reproduction were: mean total young ones per female, mean brood size, time to first reproduction, mean number broods per female and intrinsic rate of natural increase (r). Survival and growth (body length) were also determined after 21 days of exposure to the herbicide. Reproduction was significantly reduced when propanil concentration increased in the medium. The intrinsic rate of natural increase (r) decreased with increasing concentrations of propanil especially in those animals exposed to 0.55 mg/l. However, growth as well as survival of the exposed organisms only decreased in daphnids exposed to the highest propanil concentration tested. The maximum acceptable toxicant concentration (MATC) was calculated for D. magna exposed to the herbicide using as parameter of evaluation the intrinsic rate of natural increase (r). The interpolation of these results gave MATC values of 0.08 mg/l herbicide. We have derived the EC50 values for some selected parameters on D. magna exposed to propanil. EC50 values indicated that reproductive parameters were very sensitive of the effect of propanil on daphnids. Finally, the daphnids were exposed to the same sublethal herbicide concentrations as in the chronic study and the effect of the toxicant on filtration and ingestion rates was determined. Feeding rates of D. magna declined with increasing propanil concentrations. The effective propanil concentrations at which feeding rates were reduced to 50% of that in controls (EC50) were also calculated.     

Effects of lindane on Daphnia magna during chronic exposure

Abstract

Acute and chronic toxicity tests with lindane were conducted on Daphnia magna. The 24‐hr static LC50 was 1.64 mgL^‐1. The sublethal effects of 0.16, 0.25, 0.32, 0.60 and 0.80 mgL^‐1 lindane on the survival, reproduction and growth of D. magna were monitored for 21 days. The algae Nannochloris oculata (5 × 10⁵ cellsmL^‐1) was used to feed the daphnids. The parameters used to determined the effect of the pesticide on D. magna were:mean total young per female, mean brood size, days to first brood, intrinsic rate of natural increase (r), growth, and survival. Reproduction as well as survival was significantly reduced at lindane concentrations of 0.25 mgL^‐1 and higher. The intrinsic rate of natural increase (r) decreased with increasing concentrations of lindane. Growth, as measured by body length, was depressed significantly at 0.25 mgL^‐1 lindane and higher. The chronic data was used to formulate an acute/chronic ratio.     

Evaluation of a Daphnia magna renewal life‐cycle test method with diazinon

Abstract

Acute and chronic toxicity tests with diazinon (diethyl 2‐isopropyl‐6‐methyl‐4‐pyrimidinyl phosphorothionate) were conducted on Daphnia magna. The 24‐hr static LC50 was 0.86 μL.L^‐1. The sublethal effects of 0.05, 0.1, 0.5, 0.75 and 1.0 ngL^‐1 of diazinon concentrations on the survival, reproduction and growth of D. magna were monitored for 21 days. The algae Nannochloris oculata (5 x 10⁵ cellsmL^‐1) was used to feed the daphnids. The parameters used to determined the effect of the pesticide on D. magna were: mean total young per female; mean brood size; days to first brood; intrinsic rate of natural increase (r); growth; and survival. Reproduction as well as survival was significantly reduced at diazinon concentrations of 0.10 ngL^‐1 and higher. The intrinsic rate of natural increase (r) decreased with increasing concentrations of diazinon. Growth, as measured by body length, was depressed significantly at 0.05 ngL^‐1 of diazinon and higher concentrations. The maximum acceptable toxicant concentration (MATC) was calculated. The chronic data was used to formulate an acute/chronic ratio.     

Fluoxetine effects assessment on the life cycle of aquatic invertebrates

Fluoxetine is a serotonin re-uptake inhibitor, generally used as an antidepressant. It is suspected to provoke substantial effects in the aquatic environment. This study reports the effects of fluoxetine on the life cycle of four invertebrate species, Daphnia magna, Hyalella azteca and the snail Potamopyrgus antipodarum exposed to fluoxetine spiked-water and the midge Chironomus riparius exposed to fluoxetine-spiked sediments. For D. magna, a multi-generational study was performed with exposition of newborns from exposed organisms. Effects of fluoxetine could be found at low measured concentrations (around 10mugl(-1)), especially for parthenogenetic reproduction of D. magna and P. antipodarum. For daphnids, newborns length was impacted by fluoxetine and the second generation of exposed individuals showed much more pronounced effects than the first one, with a NOEC of 8.9mugl(-1). For P. antipodarum, significant decrease of reproduction was found for concentrations around 10mugl(-1). In contrast, we found no effect on the reproduction of H. azteca but a significant effect on growth, which resulted in a NOEC of 33mugl(-1), expressed in nominal concentration. No effect on C. riparius could be found for measured concentrations up to 59.5mgkg(-1). General mechanistic energy-based models showed poor relevance for data analysis, which suggests that fluoxetine targets specific mechanisms of reproduction.     

Multi-generational effects of four selected environmental oestrogens on Daphnia magna

The objective of this study was to determine whether vertebrate-type oestrogens have ecotoxicological effects on a crustacean species. The effects of 17beta-oestradiol (E2), diethylstilbestrol (DES), bisphenol A (BPA) and 4-nonylphenol (4-NP) on the freshwater invertebrate Daphnia magna were assessed over first and second generations. The acute EC(50) 48 h, based on immobilisation, for E2, DES, BPA and 4-NP were 2.87 mg/l, 1.55 mg/l, 7.75 mg/l and 0.13 mg/l, respectively. The impact of the test chemicals on moulting frequency was also assessed. The EC(50) 48 h, based on the inhibition of moult number for E2, DES and 4-NP were 2.04 mg/l, 1.87 mg/l and 0.14 mg/l, respectively. BPA was not observed to impact the moulting frequency of D. magna at concentrations tested. In a series of separate studies, the effects of the four selected test compounds on the survival, moulting frequency and reproduction of first and second generational D. magna were assessed over a period of 21 d. Exposure of D. magna to 4-NP decreased the number of offspring produced in both first and second generation testing. DES proved to have no significant (p0.05) inhibition of fecundity in first generation but when second generation daphnids were exposed to DES, a significant (p0.05) reduction in the number of offspring was recorded. When D. magna were exposed to E2 or BPA, no statistically significant (p0.05) inhibition in the number of moults or offspring produced was observed.     

An assessment of the chronic toxicity of the synthetic pyrethroid, fenvalerate, to Daphnia galeata mendotae, using life tables

The effects of chronic exposure to fenvalerate, a synthetic pyrethroid insecticide, on the cladoceran Daphnia galeata mendotae were evaluated under laboratory conditions. The daphnids were exposed for their entire life cycle to concentrations ranging from 0.005 to 0.1 microg fenvalerate litre(-1). The parameters used to determined toxicity were survival, time to first reproduction, reproductive frequency, number of young per brood, cumulative brood size, intrinsic rate of natural increase (r(m)), generation time (T) and net reproductive rate (R(0)). A concentration of 0.005 microg fenvalerate litre(-1) resulted in an increase in longevity but a decrease in production of young. Higher concentrations caused a decrease in survival. Studies of shorter duration than the complete life cycle of the organisms would not have detected toxicity at such low levels. The intrinsic rate of natural increase, r(m), was not affected by fenvalerate until concentration reached 0.05 microg litre(-1) and r(m) decreased to 0.113 from 0.238. A concentration of 0.01 microg litre(-1) caused the net reproductive rate, R(0), and the generation time, T, to decrease to 73.2 offspring per female and 17.3 days from 125.9 offspring per female and 20.3 days, respectively.     

Effects of thiobencarb herbicide to an alga (Nannochloris oculata) and the cladoceran (Daphnia magna)

Chronic toxicity studies were conducted with an algae (Nannochloris oculata) and the cladoceran (Daphnia magna) to determine their relative sensitivities to the thiocarbamate herbicide thiobencarb (S-4-chlorobenzyl diethylthiocarbamate). Most of the algal populations were initially affected by exposure to the herbicide. Thiobencarb concentrations higher than 0.5 mg/L significantly reduced algal densities after 24-h exposure. The 24-h static EC50 in D. magna was 3.01 mg/L. The sublethal effects of 0.3, 0.37, 0.5, 0.75, and 1.5 mg/L of thiobencarb concentrations on the survival, reproduction, and growth of D. magna were monitored for 21 days. The parameters used to determined the effect of the herbicide on D. magna were mean total young per female; mean brood size; days to first brood; intrinsic rate of natural increase (r); growth; and survival. Reproduction was significantly reduced at thiobencarb concentrations of 0.30 mg/L and higher while survival was affected after exposure to 0.75 and 1.5 mg/L of the pesticide. The r value decreased with increasing concentrations of thiobencarb. Growth, as measured by body length, was depressed significantly after exposure to all herbicide concentrations tested.     

Juvenile hormone agonists affect the occurrence of male Daphnia

The water flea Daphnia magna reproduces primarily by cyclic parthenogenesis. Environmental stimuli that signal a change to adverse conditions induce the organisms to switch from parthenogenesis to gamogenetic reproduction. During the gamogenetic period, they produce male daphnids and dormant resting eggs, which can survive prolonged periods of environmental adversity. However, little is known about the mechanisms associated with the switch from parthenogenesis to gamogenetic reproduction. We investigated the effects of several juvenoids on sex determination in Daphnia. Females less than 24 h old were exposed to various concentrations of the test substance and were observed for 21 days. It was found that they can trigger the appearance of male daphnids: the percentage of males in the population increases to a level greater than what occurs under ordinary environmental conditions. We found that methylfarnesoate, juvenile hormone III, methoprene, and the phenoxyphenoxy derivatives pyriproxyfen and fenoxycarb (both insecticides) reduced the production of offspring and produced sex ratios dominated by male daphnids. Pyriproxyfen and fenoxycarb showed striking effects at low concentrations. Exposure to either of these chemicals at a concentration of 330 ngl(-1) caused adult females to produce almost all male neonates. Methylfarnesoate, juvenile hormone III, and methoprene showed an effect in inducing male production at higher concentrations (3.7 x 10(3), 3.3 x 10(5), and 1.3 x 10(5) ngl(-1), respectively). Our findings suggest that juvenile hormone agonists, including some insecticides, affect the chemical signaling responsible for inducing the production of male offspring.     

Multi-generation cadmium acclimation and tolerance in Daphnia magna Straus

The cladoceran Daphnia magna was acclimated for seven generations to cadmium concentrations ranging from 0 (control) to 250 microg/l Cd (corresponding to a free ion activity of 4.60 nM Cd2+). Acute and chronic cadmium tolerance as well as cadmium accumulation were monitored as a function of acclimation time. After two to three generations of acclimation to concentrations ranging from 0.23 to 1.11 nM Cd2+ increases in acute tolerance were maximal (factor 7.2) and significant. Acclimation for seven generations to the same acclimation concentrations did result in an increased chronic cadmium tolerance (21 days EC50 values increased). Organisms acclimated to 1.93 nM Cd2+ were equally or more sensitive than non-acclimated daphnids in acute and chronic toxicity tests. Cadmium contents in D. magna increased significantly as a function of the acclimation concentration. Maximum body burdens of 236+/-30 microg Cd/g dry weight were measured in organisms exposed to 4.60 nM Cd2+, but detoxification mechanisms were only successful up to 82+/-20 microg Cd/g dry weight as this concentration did not cause major decreases in survival and reproduction in chronic toxicity tests. As the potential positive effect of acclimation on cadmium tolerance disappeared with successive acclimation generations and increasing acclimation concentrations, it is concluded that multi-generation acclimation studies are important for the evaluation of the long-term effects of environmental toxicants.     

The influence of feeding, photoperiod and selected solvents on the reproductive strategies of the water flea, Daphnia magna

The influence of different feeding rates, reduced photoperiod and ethanol and acetone exposure on male production in Daphnia magna was investigated. Male production was observed under reduced photoperiod (8-h light:16-h dark) in all food levels. However, the sex ratio of daphnids fed 1x10(6) algae cells increased significantly from that of daphnids fed 15x10(6) algae cells (0.486+/-0.059 and 0.271+/-0.027, respectively). Under normal photoperiod (16-h light:8-h dark) and low food conditions (3.75x10(6) algae cells), a statistically significant increase in the sex ratio was observed in daphnids exposed to acetone at EPA acceptable solvent limits (0.1 ml/l). A shift back to parthenogenetic reproduction was observed in acetone-exposed daphnids at high food levels (15x10(6) algae cells). The results indicate that the reproductive strategies of females are influenced by phenological (i.e. reduced photoperiod), dietary and exogenous chemical cues.     

Copper regulation and homeostasis of Daphnia magna and Pseudokirchneriella subcapitata: influence of acclimation

This study aimed to evaluate (1) the capacity of the green alga Pseudokirchneriella subcapitata and the waterflea Daphnia magna to regulate copper when exposed to environmentally realistic copper concentrations and (2) the influence of multi-generation acclimation to these copper concentrations on copper bioaccumulation and homeostasis. Based on bioconcentration factors, active copper regulation was observed in algae up to 5 microg Cu L(-1) and in daphnids up to 35 mug Cu L(-1). Constant body copper concentrations (13+/-4 microg Cu g DW(-1)) were observed in algae exposed to 1 through 5 microg Cu L(-1) and in daphnids exposed to 1 through 12 microg Cu L(-1). At higher exposure concentrations, there was an increase in internal body copper concentration, while no increase was observed in bioconcentration factors, suggesting the presence of a storage mechanism. At copper concentrations of 100 microg Cu L(-1) (P. subcapitata) and 150 microg Cu L(-1) (D. magna), the significant increases observed in body copper concentrations and in bioconcentration factors may be related to a failure of this regulation mechanism. For both organisms, internal body copper concentrations lower than 13 microg Cu g DW(-1) may result in copper deficiency. For P. subcapitata acclimated to 0.5 and 100 microg Cu L(-1), body copper concentrations ranged (mean+/-standard deviation) between 5+/-2 microg Cu g DW(-1) and 1300+/-197 microg Cu g DW(-1), respectively. For D. magna, this value ranged between 9+/-2 microg Cu g DW(-1) and 175+/-17 microg Cu g DW(-1) for daphnids acclimated to 0.5 and 150 microg Cu L(-1). Multi-generation acclimation to copper concentrations >or =12 microg Cu L(-1) resulted in a decrease (up to 40%) in body copper concentrations for both organisms compared to the body copper concentration of the first generation. It can be concluded that there is an indication that P. subcapitata and D. magna can regulate their whole body copper concentration to maintain copper homeostasis within their optimal copper range and acclimation enhances these mechanisms.     

Comparative toxicity of rac- and S-tebuconazole to Daphnia magna

Tebuconazole is a chiral triazole fungicide used as raceme in a variety of agricultural applications. Earlier studies showed that tebuconazole is toxic to many non-target aquatic organisms but relative data for tebuconazole enantiomers are lacking. Thus, goal of this study was to evaluate and compare the toxicity of rac- and S-tebuconazole with Daphnia magna at both acute and chronic levels according to Organization for Economic Cooperation and Development (OECD) guidelines 202 and 211 respectively, to provide some guidelines for optimizing chiral pesticides application and management. The exposure concentrations were 0.1, 0.5, 1, 2, 4, 8, 10 mg L^?1 for both rac- and S-tebuconazole and their 48-h EC₅₀ values to D. magna were 3.53 (3.32–3.78) and 2.74 (2.33–3.10) mg L^?1 respectively, indicating that these both are medium toxic to D. magna with no significant toxicity difference at acute level. In chronic test, <24-h old D. magna were exposed to 0.01, 0.05, 0.10, 0.20, and 0.40 mg L^?1 of rac- and S-tebuconazole with one blank and one solvent control for 21 days according to OECD guideline 211. Four developmental (molting rate, days to the 1st and 3rd brood, and body length) and five reproductive (size of the 1st and 3rd brood, number of broods, and number of neonates) parameters for each D. magna were determined. Results showed that both rac- and S-tebuconazole significantly reduced the reproduction and impacted the development of D. magna at concentrations of 0.05 mg L^?1 or higher. Furthermore, S-tebuconazole was more toxic than raceme, and the difference between effects on the same parameters induced by rac- and S-tebuconazole was statistically significant. These results demonstrated that the chronic toxicity of S-tebuconazole might be underestimated in general use, and further studies should focus more on the biological behaviors of enantiomers and not just the raceme of tebuconazole and other chiral pesticides in the environment.     

Characterizing the toxicity of pulsed selenium exposure to Daphnia magna

The acute toxicity of selenium (Se) to aquatic biota has been studied extensively for decades. However, most studies have used a constant concentration aqueous exposure of Se to an invertebrate species. Since constant concentration exposure of toxicants to invertebrates is unusual in the environment, episodic exposure or pulsed exposures may represent true risk to aquatic biota more accurately. This research was designed to characterize the toxicity effects of pulsed Se exposure to Daphnia magna. Selenium exposure was varied during a 21-d chronic toxicity test to examine the effects of exposure concentration, duration, and recovery on survival, growth, and reproduction of D. magna. While D. magna did not die during exposures, latent mortality was observed. Latent mortality increased with exposure concentration and duration. Hence, standard toxicity test using continuous exposures would underestimate Se toxicity. Risk assessment method using results of continuous exposure would underestimate risk of Se to biota. For double-pulse exposures, cumulative mortality on day 21 was higher when time interval between pulses was shorter. With the same total exposure time, continuous exposure caused higher toxicity than did pulsed exposures due to recovery and tolerance development in D. magna after earlier pulses. Growth and reproduction of surviving D. magna were not affected by pulsed Se exposure due to recovery of D. magna after removal of the pulses. Based on these results, risk assessment for Se should take latent effects and the effect of recovery in to account.     

Effects of phytosterols on zebrafish reproduction in multigeneration test

Zebrafish from mixed sex populations were exposed continuously across three generations to two phytosterol preparations both containing -sitosterol. The phytosterols were isolated from wood and soy beans. Blood vitellogenin levels and sex ratio changes were used as intermediate indicators of the reproduction failures. Both sterol preparations caused vitellogenin induction in the exposed fish. The wood sterol changed the sex ratio of the exposed fish. In generation F1, the predominant sex was male, and in generation F2 it was female. The soy sterol in the used test concentration was lethal to the exposed fish in generation F1. This multigeneration test evidenced that phytosterols containing -sitosterol disrupt the reproduction system of zebrafish by changing the sex ratios and by inducing the vitellogenin production in the exposed fish.     

Effects of pharmaceuticals on Daphnia survival, growth, and reproduction

Pharmaceuticals have been globally detected in surface waters, and the ecological impacts of these biologically-active, ubiquitous chemicals are largely unknown. To evaluate the aquatic toxicity of individual pharmaceuticals and mixtures, we performed single species laboratory toxicity tests with Daphnia magna, a common freshwater zooplankton. We conducted acute (6-day) and chronic (30-day) exposure pharmaceutical bioassays and evaluated survivorship and morphology of adults and neonates, adult length, resting egg production, brood size (fecundity), and the proportion of male broods produced (sex ratio). In general, exposure to a single pharmaceutical in the 1-100 microg/l range yielded no apparent effects on the normal life processes of Daphnia. However, chronic fluoxetine exposure (36 microg/l) significantly increased Daphnia fecundity, and acute clofibric acid exposure (10 microg/l) significantly increased sex ratio. A mixture of fluoxetine (36 microg/l) and clofibric acid (100 microg/l) caused significant mortality; the same fluoxetine concentration mixed with 10 microg/l clofibric acid resulted in significant deformities, including malformed carapaces and swimming setae. Mixtures of three to five antibiotics (total antibiotic concentration 30-500 microg/l) elicited changes in Daphnia sex ratio. We conclude: (1) individual and mixtures of pharmaceuticals affect normal development and reproduction of Daphnia magna, (2) aquatic toxicity of pharmaceutical mixtures can be unpredictable and complex compared to individual pharmaceutical effects, and (3) timing and duration of pharmaceutical exposure influence aquatic toxicity.     

Ecological impact assessment of metallurgic effluents using in situ biomarker assays

An ecological impact study was performed based on in situ biomarker assays with the waterflea Daphnia magna. The effects of metallurgic effluents on the energy metabolism, anti-oxidative metabolism and DNA damage were assessed in caged daphnids during a 4-week study. In situ survival and reproduction studies demonstrated a clear impact on these parameters in organisms exposed in the most polluted areas. At the downstream--sublethal--zone the organisms were disturbed within their tolerance limits, resulting in alterations of their energy metabolism. These data suggest an acclimation hypothesis, which was tested through the analysis of the energy metabolism of resident species: isopods and amphipods. These organisms had shifted to a decrease in their overall energy metabolism compared to the upstream region. This change in some biochemical processes suggests a selective advantage to cope with the prevailing environmental conditions. In addition, we found clear genotoxic effects caused by the industrial discharges that might correlate with a reduction in (long-term) survival.     

Multigenerational cadmium acclimation and biokinetics in Daphnia magna

A Cd exposure (3 microg L(-1)) experiment was conducted for six successive generations to investigate the responses to chronic Cd stress in Daphnia magna. We observed a biphasic accumulation of Cd in the six generations and suggested a similar pattern with respect to daphnids' tolerance. Cd assimilation efficiencies, daphnid growth, and reproduction corresponded to the changes of tolerance, which was partially accounted for by metallothionein induction. When maternally exposed neonates grew in Cd-free water for one or two generations, their growth, MT concentration and biokinetic parameters partially or totally recovered. The rapid recovery suggests the high potential for ecological restoration from Cd pollution. Our results indicate that the tolerance of sensitive D. magna clones to Cd was dependent on long-term or multigenerational exposure. The tolerance developed within the first several generations might not be maintained, and the animals may become even more sensitive to Cd stress in subsequent generations.     

Evaluation of the Daphnia magna reproduction test for detecting endocrine disruptors

The Daphnia 21 d reproduction test is considered as a comprehensive and decisive test in the OECD Conceptual Framework for testing and assessment of endocrine disrupting chemicals (EDCs). However, how to interpret results of the Daphnia 21 d reproduction test for identification, risk assessment and testing strategy of EDCs remains an unsolved issue. This study analysed a total number of 135 published studies encompassing 86 known EDCs and non-EDCs with different modes of action. Our results show that the majority of effects on apical endpoints (survival, molting, growth, time to reproductive maturity, brood size, the number of broods, and the total number of offspring) do not seem to be EDC-specific. In contrast, the endpoint sex ratio is likely specific to juvenile hormones and their mimics. Variability is quantified for three most reported endpoints survival, the total number of offspring and sex ratio. Quantification of the endpoint sensitivity shows that the sensitivity of the sex ratio is lower than that of the total number of offspring. The Daphnia 21 d reproduction test gives insufficient information to conclude if a substance is an EDC or not. EDCs that are potent in assays in vitro may not be potent in the Daphnia 21 d reproduction test. We conclude that the Daphnia 21 d reproduction test is important for deriving No Observed Effect Concentrations for risk assessment but may produce false negatives in identification of EDCs when used on its own. A targeted testing strategy for selection of species, tests, and endpoints is suggested for identifying EDCs.     

Production of male neonates in Daphnia magna (Cladocera, Crustacea) exposed to juvenile hormones and their analogs

We exposed the water flea Daphnia magna (Cladocera, Crustacea) to either juvenile hormone I (JH I), juvenile hormone II (JH II), or the juvenile hormone-mimicking insecticides kinoprene, hydroprene, epofenonane, or fenoxycarb. By 21-day reproduction tests, we investigated the effects on the number of neonates born per female and the offspring sex ratio. All six chemicals induced D. magna to produce male neonates; the male sex ratio of the offspring increased as the chemical concentration increased. EC50 values for production of male neonates were estimated as 400 (JH I), 410 (JH II), 190 (kinoprene), 2.9 (hydroprene), 64 (epofenonane), and 0.92 (fenoxycarb) microg/l. The number of neonates produced was reduced with all chemicals at the concentrations investigated. At the EC50 for male production, five of the six chemicals reduced the reproductive rate to less than 50%; the exception was epofenonane, which caused only a slight reduction in reproductive rate. These results were similar to those obtained for five juvenoids studied previously, one of which was studied here again. There are now 10 chemical substances--all juvenile hormones or their analogs-that are known to induce D. magna to produce male neonates. This suggests that juvenile hormone is involved in initiating male production followed by sexual reproduction in D. magna, and probably in most cladocerans that exhibit cyclic parthenogenesis.