Demographic Assessment of Mono(2-ethylhexyl) Phthalate (MEHP) and Monoethyl Phthalate (MEP) Concentrations in Common Bottlenose Dolphins (Tursiops truncatus) From Sarasota Bay, FL, USA.

Common bottlenose dolphins (Tursiops truncatus) have previously demonstrated exposure to phthalate esters. Phthalates and phthalate esters are commonly added to consumer goods to enhance desirable properties. As the amount of plastic marine debris increases, these chemicals can easily leach from these products into the surrounding environment. To evaluate demographic variability in exposure, eight phthalate metabolites were quantified in urine samples collected from free-ranging bottlenose dolphins sampled in Sarasota Bay, FL, USA (2010-2019; n = 51). Approximately 75% of individual dolphins had detectable concentrations of at least one phthalate metabolite. The most frequently detected metabolites were mono(2-ethylhexyl) phthalate (MEHP; n = 28; GM = 4.57 ng/mL; 95% CI = 2.37-8.80; KM mean = 7.95; s.d. = 15.88) and monoethyl phthalate (MEP; GM = 4.51 ng/mL; 95% CI = 2.77-7.34; ROS mean = 2.24; s.d. = 5.58). Urinary concentrations of MEHP and MEP were not significantly different between sex (MEHP p = 0.09; MEP p = 0.22) or age class (i.e., calf/juvenile vs. adult; MEHP p = 0.67; MEP p = 0.13). Additionally, there were no significant group differences in the likelihood of MEHP or MEP detection for any demographic as determined by a Peto-Peto test. Frequency of detection was similar for both metabolites between males and females (MEHP p = 0.10; MEP p = 0.40) as well as between juveniles and adults (MEHP p = 0.50; MEP: p = 0.60). These findings suggest ubiquitous exposure risk for both sexes and age classes, warranting further investigation into potential sources and health implications.

Pathologic findings and causes of death in bottlenose dolphins Tursiops truncatus stranded along the Georgia coast, USA (2007-2013).

Between 2007 and 2013, before the 2013 cetacean morbillivirus outbreak, 26 fresh bottlenose dolphin carcasses were necropsied on the coast of Georgia, USA. Here, we present the pathological and microbiological findings associated with their most likely causes of death. The primary cause of death was determined in 25 individuals and included systemic bacterial infection (n = 7), verminous and bacterial bronchopneumonia (n = 5), drowning/entanglement (n = 5), disseminated histoplasmosis (n = 1), intestinal intussusception (n = 1), vegetative endocarditis (n = 1), meningitis (n = 1), necrotizing dermatitis (n = 1), disseminated angiomatosis (n = 1), emaciation (n = 1) and stingray spine trauma (n = 1). Histiocytic and eosinophilic bronchopneumonia associated with Halocerchus sp. infection was observed in 69% of the animals (18/26) and eosinophilic gastritis due to Anisakidae nematodes was found in 36% of the examined stomachs (8/22). Moderate to severe eosinophilic pancreatitis with fibrosis was observed in 4 animals infected with Brachycladiidae trematodes. Proliferative and ulcerative lymphoplasmacytic dermatitis was found in 5 animals and was considered to contribute to deteriorated health status in 2 calves. Pulmonary and lymph node angiomatosis were observed in 15 and 10 animals, respectively. In at least 2 animals, the concentration of polychlorinated biphenyls (PCBs) in the blubber exceeded 1500 µg g-1 of lipid. Bottlenose dolphins stranded on the Georgia coast have a wide range of inflammatory lesions associated with a variety of helminth, bacterial, and fungal pathogens. Some resident animals have also been exposed to high levels of PCB contamination, which could reduce host immunocompetence. Higher exposure to these or other pathogens could result in further decline in the health of resident and migrant dolphin populations in this region.

Comparative Toxicity of Two Chemical Dispersants and Dispersed Oil in Estuarine Organisms.

Chemical dispersants can be a useful tool to mitigate oil spills. This study examined potential risks to sensitive estuarine species by comparing the toxicity of two dispersants (Corexit® EC9500A and Finasol® OSR 52) individually and in chemically enhanced water-accommodated fractions (CEWAFs) of Louisiana Sweet Crude oil. Acute toxicity thresholds and sublethal biomarker responses were determined in seven species (sheepshead minnow, grass shrimp, mysid, amphipod, polychaete, hard clam, mud snail). Comparing median lethal (LC50) values for the dispersants, Finasol was generally more toxic than Corexit and had greater sublethal toxicity (impaired embryonic hatching, increased lipid peroxidation, decreased acetylcholinesterase activity). The nominal concentration-based mean LC50 for all species tested with Corexit was 150.31 mg/L compared with 43.27 mg/L with Finasol. Comparing the toxicity of the CEWAFs using the nominal concentrations (% CEWAF), Corexit-CEWAFs appeared more toxic than Finasol-CEWAFs; however, when LC50 values were calculated using measured hydrocarbon concentrations, the Finasol-CEWAFs were more toxic. There was greater dispersion efficiency leading to greater hydrocarbon concentrations measured in the Corexit-CEWAF solutions than in equivalent Finasol-CEWAF solutions. The measured concentration-based mean LC50 values for all species tested with Corexit-CEWAF were 261.96 mg/L total extractable hydrocarbons (TEH) and 2.95 mg/L total polycyclic aromatic hydrocarbons (PAH), whereas the mean LC50 values for all species tested with Finasol-CEWAF were 23.19 mg/L TEH and 0.49 mg/L total PAH. Larval life stages were generally more sensitive to dispersants and dispersed oil than adult life stages within a species. These results will help to inform management decisions regarding the use of oil-spill dispersants.

High Variability of Insulin Sensitivity in Closely Related Obese Mouse Inbred Strains.

Obesity is one of several risk factors for insulin resistance and type 2 diabetes. Here we examined males of 6 obese mouse inbred lines derived from the Berlin Fat Mouse (BFM) outbred population with respect to insulin sensitivity and factors of the metabolic syndrome with focus on the skeletal muscle as a major target of insulin dependent glucose uptake.Males were kept on a rodent standard diet and several approaches were carried out to address insulin sensitivity, adiposity and lipids in the serum. Transcript and protein levels of several genes in the insulin signalling pathway were measured. 2 of the lines, BFMI860-12 and in particular BFMI861-S1, showed a markedly reduced insulin sensitivity already at the age of 20 weeks. BFMI861-S1 mice also displayed elevated liver triglyceride levels as a sign of lipid overload and ectopic fat storage. The analysis of the insulin signalling pathway in skeletal muscle provided evidence for low insulin receptor (INSR) and normal glucose 4 transporter (GLUT4) protein amounts in BFMI861-S1 mice, while BFMI860-12 mice showed increased INSR and very low GLUT4 protein amounts. Interestingly, the sublines BFMI860-S2 and BFMI861-S2, which are highly related to the former 2 lines, respectively, were inconspicuously insulin sensitive. The expected few genetic differences among the BFMI lines facilitate the identification of causal genetic variation. This study identified 2 mouse lines with different impairments of insulin signalling. These lines resemble useful models for studying mechanisms leading to the pathophysiology of the metabolic syndrome, in particular insulin resistance.

Impacts of Coastal Development on the Ecology of Tidal Creek Ecosystems of the US Southeast including Consequences to Humans.

Upland areas of southeastern U.S. tidal creek watersheds are popular locations for development, and they form part of the estuarine ecosystem characterized by high economic and ecological value. The primary objective of this work was to define the relationships between coastal development, with its concomitant land use changes and associated increases in nonpoint source pollution loading, and the ecological condition of tidal creek ecosystems including related consequences to human populations and coastal communities. Nineteen tidal creek systems, located along the southeastern United States coast from southern North Carolina to southern Georgia, were sampled during summer, 2005 and 2006. Within each system, creeks were divided into two primary segments based upon tidal zoning: intertidal (i.e., shallow, narrow headwater sections) and subtidal (i.e., deeper and wider sections) and then watersheds were delineated for each segment. Relationships between coastal development, concomitant land use changes, nonpoint source pollution loading, the ecological condition of tidal creek ecosystems, and the potential impacts to human populations and coastal communities were evaluated. In particular, relationships were identified between the amount of impervious cover (indicator of coastal development) and a range of exposure and response measures including increased chemical contamination of the sediments, increased pathogens in the water, increased nitrate/nitrite levels, increased salinity range, decreased biological productivity of the macrobenthos, alterations to the food web, increased flooding potential, and increased human risk of exposure to pathogens and harmful chemicals. The integrity of tidal creeks, particularly the headwaters or intertidally-dominated sections, were impaired by increases in nonpoint source pollution associated with sprawling urbanization (i.e., increases in impervious cover). This finding suggests these habitats are valuable early warning sentinels of ensuing ecological impacts and potential public health and flooding risk from sprawling coastal development. Results also validate the use of a conceptual model with impervious cover thresholds for tidal creek systems in the southeast region.

Distribution and sources of PCBs (Aroclor 1268) in the Sapelo Island National estuarine research reserve.

Aroclor 1268 is a highly chlorinated PCB mixture that was released into the aquatic environment near Brunswick, GA (BR), as a result of decades of local industrial activity. This extensive contamination has led to US EPA Superfund designation in estuarine areas in and around Purvis Creek, GA. Roughly 50 km to the northeast is the Sapelo Island National Estuarine Research Reserve (SI) where previous studies have documented unexpectedly high Aroclor 1268-like PCB levels in blubber and plasma samples of resident bottlenose dolphins. This result led to a collaborative effort to assess the PCB patterns and concentrations in SI sediment and fish (as potential vectors for PCB transfer to SI resident dolphins). Thirty SI randomly assigned stations were sampled for sediment PCB levels. Additionally, fish were collected and analyzed from SI (n = 31) and BR (n = 33). Results were pooled with regional assessments of PCB concentrations from South Carolina and North Carolina in an effort to determine the association of Aroclor 1268 levels in SI samples. Results indicated that PCB levels in sediment and fish are much lower in the SI estuary compared to BR sediment and fish concentrations. However, PCB congener profiles for both sediments and fish were similar between the two locations and consistent with the Aroclor 1268 signature, indicating possible transport from the Brunswick area. A likely source of Aroclor 1268 in dolphins from SI is contaminated fish prey.

Synergistic targeting/prodrug strategies for intravesical drug delivery--lectin-modified PLGA microparticles enhance cytotoxicity of stearoyl gemcitabine by contact-dependent transfer.

The direct access to the urothelial tissue via intravesical therapy has emerged as a promising means for reducing the high recurrence rate of bladder cancer. However, few advanced delivery concepts have so far been evaluated to overcome critical inherent efficacy limitations imposed by short exposure times, low tissue permeability, and extensive washout. This study reports on a novel strategy to enhance gemcitabine treatment impact on urothelial cells by combining a pharmacologically advantageous prodrug approach with the pharmacokinetic benefits of a glycan-targeted carrier system. The conversion of gemcitabine to its 4-(N)-stearoyl derivative (GEM-C18) allowed for stable, homogeneous incorporation into PLGA microparticles (MP) without compromising intracellular drug activation. Fluorescence-labeled GEM-C18-PLGA-MP were surface-functionalized with wheat germ agglutinin (WGA) or human serum albumin (HSA) to assess in direct comparison the impact of biorecognitive interaction on binding rate and anchoring stability. MP adhesion on urothelial cells of non-malignant origin (SV-HUC-1), and low- (5637) or high-grade (HT-1376) carcinoma was correlated to the resultant antiproliferative and antimetabolic effect in BrdU and XTT assays. More extensive and durable binding of the WGA-GEM-C18-PLGA-MP induced a change in the pharmacological profile and substantially higher cytotoxicity, allowing for maximum response within the temporal restrictions of instillative administration (120 min). Mechanistically, a direct, contact-dependent transfer of stearoyl derivatives from the particle matrix to the urothelial membrane was found to account for this effect. With versatile options for future application, our results highlight the potential offered by the synergistic implementation of targeting/prodrug strategies in delivery systems tailored to the intravesical route.

Potential consequences of the Fukushima accident for off-site nuclear emergency management: a case study for Germany.

The Fukushima accident led to high radionuclide releases into the atmosphere for more than 3 weeks. This situation has not been assumed when the concepts of nuclear emergency preparedness were developed internationally. The results of simulations studying potential implications of Fukushima-like source terms on nuclear emergency preparedness are presented. Two hypothetical source terms are considered. Radiological consequences are assessed with the decision support system RODOS. Atmospheric dispersion calculations are based on meteorological monitoring data from June and December 2010, respectively, to study potential seasonal effects. Simulations are performed for two nuclear power plant sites in Northern and Southern Germany, respectively. These sites are chosen due to their differing meteorology and topography. Predicted radiation doses of members of the population are compared with dose reference levels actually recommended for initiating protective measures in Germany. Potential implications of general interest for nuclear emergency planning are discussed.

Effects of chemically spiked sediments on estuarine benthic communities: a controlled mesocosm study.

Ambient sediments were collected from a reference site in the North Edisto River, SC and transferred to a laboratory facility to investigate effects of chemical contaminants on estuarine infaunal communities under controlled mesocosm conditions. Sediment contaminant slurries were prepared using dried sediments collected from the reference site and spiked with a metal (copper), a polycyclic aromatic hydrocarbon (pyrene), and a pesticide (4,4(')-dichlorodiphenyltrichloroethane) to yield nominal mean effects range-median (ERM) quotients of <0.01 (no addition), 0.1, and 1.0 and applied to control, low dose (TRT A), and high dose (TRT B) treatment groups, respectively. Sediment samples for contaminant and benthic analyses were collected at the start of the experiment, 1 month after dosing, and 3 months after dosing. Near-nominal mean ERM quotients of 0.001, 0.075, and 0.818 were measured initially after dosing and remained fairly constant throughout the experiment. Measures of benthic condition, diversity, and richness were significantly reduced in both treatment groups relative to the control 1 month after dosing and persisted in TRT B at 3 months. The results demonstrate that benthic community effects can be observed at mean ERM quotients that are about an order of magnitude lower than levels that have been shown to be associated with significant toxicity in acute laboratory bioassays with single species (e.g., amphipods) in other studies.

Effects of fluoxetine exposure on serotonin-related activity in the sheepshead minnow (Cyprinodon variegatus) using LC/MS/MS detection and quantitation.

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor and is among the top 100 drugs prescribed yearly in the United States and the United Kingdom. Tissue and water extraction methods were developed to detect and quantify FLX, norfluoxetine and the associated biological compounds serotonin (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), tryptophan (TRP) and melatonin (MEL) using LC/MS/MS. Acute mortality and sublethal physiological effects of FLX were assessed using standard static renewal toxicity tests in which juvenile sheepshead minnows (Cyprinodon variegatus) were exposed to FLX. Fluoxetine did not cause significant mortality at levels near currently reported environmental concentrations. Significant changes in neurotransmitter levels were observed within the serotonergic system in juvenile sheepshead minnows exposed at concentrations approximately one order of magnitude above those currently reported in the environment. Transformation activity ratios of a product to a precursor compound (5-HT/TRP, 5-HIAA/5-HT and MEL/5-HT) also exhibited significant changes with FLX treatment. Fluoxetine exposure did not only affect 5-HT but had additional effects both upstream and downstream of 5-HT within its synthesis and metabolic pathways. These sublethal changes within the serotonergic pathway may result in behavioral changes which could, in turn, have implications for the ecological response of populations to additional environmental stressors.

An evaluation of contaminated estuarine sites using sediment quality guidelines and ecological assessment methodologies.

Toxic contaminants may enter estuarine ecosystems through a variety of pathways. When sediment contaminant levels become sufficiently high, they may impact resident biota. One approach to predict sediment-associated toxicity in estuarine ecosystems involves the use of sediment quality guidelines (ERMs, ERLs) and site-specific contaminant chemistry while a second approach utilizes site-specific ecological sampling to assess impacts at the population or community level. The goal of this study was to utilize an integrated approach including chemical contaminant analysis, sediment quality guidelines and grass shrimp population monitoring to evaluate the impact of contaminants from industrial sources. Three impacted sites and one reference site were selected for study. Grass shrimp populations were sampled using a push-netting approach. Sediment samples were collected at each site and analyzed for metals, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and pesticides. Contaminant levels were then compared to sediment quality guidelines. In general, grass shrimp population densities at the sites decreased as the ERM quotients increased. Grass shrimp densities were significantly reduced at the impacted site that had an ERM exceedance for chromium and the highest Mean ERM quotient. Regression analysis indicated that sediment chromium concentrations were negatively correlated with grass shrimp density. Grass shrimp size was reduced at two sites with intermediate levels of contamination. These findings support the use of both sediment quality guidelines and site-specific population monitoring to evaluate the impacts of sediment-associated contaminants in estuarine systems.

The effects of the contemporary-use insecticide (fipronil) in an estuarine mesocosm.

To examine the effects of environmentally realistic fipronil concentrations on estuarine ecosystems, replicated mesocosms containing intact marsh plots and seawater were exposed to three treatments of fipronil (150, 355, and 5000 ng/L) and a Control. Juvenile fish (Cyprinidon variegatus), juvenile clams (Mercenaria mercenaria), oysters (Crassostrea virginica), and grass shrimp (Palaemonetes pugio) were added prior to fipronil in an effort to quantify survival, growth, and the persistence of toxicity during the planned 28-day exposure. Results indicated that there were no fipronil-associated effects on the clams, oysters, or fish. Shrimp were sensitive to the highest two concentrations (40% survival at 355 ng/L and 0% survival at 5000 ng/L). Additionally, the highest fipronil treatment (5000 ng/L) was toxic to shrimp for 6 weeks post dose. These results suggest that fipronil may impact shrimp populations at low concentrations and further use in coastal areas should be carefully assessed.

Toxicological studies in tropical ecosystems: an ecotoxicological risk assessment of pesticide runoff in South Florida estuarine ecosystems.

A multiyear study in the C-111 canal system and associated sites in Florida Bay was undertaken to determine the potential pesticide risk that exists in South Florida. After the examination of extensive pesticide concentration data in surface water, tissues, and semipermeable membrane devices (SPMDs), canal contamination seems to be derived from the extensive agricultural production that drains into the C-111 canal. The results of this study indicate that runoff from agricultural processes led to quantifiable pesticide residues in both canal and bay surface water, which occasionally exceeded current water quality criteria. The major pesticide of concern was endosulfan, which was detected at 100% of the sites sampled. Endosulfan exposure did not cause any acute effects in fish and crustaceans deployed in field bioassays. Chronic effects were observed in copepods, clams, and oysters but could not be attributed to endosulfan exposure. The decision to alter the C-111 canal flow and allow increased freshwater flow into the adjacent Everglades National Park may result in discharges of pesticides into the Everglades. Continued monitoring in this area is needed during this change in flow regime.

Reproductive alterations in adult grass shrimp, Palaemonetes pugio, following sublethal, chronic endosulfan exposure.

Grass shrimp (Palaemonetes pugio) populations exposed to anthropogenic contaminant sources in South Carolina (SC) have reduced densities when compared with populations at SC-reference sites. This laboratory study examined the effects of a commonly used agricultural insecticide, endosulfan, on grass shrimp reproduction. Reproductively active grass shrimp were chronically exposed to sublethal concentrations of endosulfan (200 or 400 ng/l) for 43 days. The cumulative number of females that became gravid and the rate at which they became gravid were measured. Endosulfan exposure reduced the cumulative number of gravid females by 31% in the 200 ng/l exposure and 39% in the 400 ng/l exposure. The first appearance of gravid females in the population was significantly delayed in treated populations compared with the control treatment in a dose dependent manner. Clutch size in these gravid females was not significantly different among the treatments. Additionally, there was no difference in the onset of reproduction in the treated populations. These results implicate a population reduction due to a decrease in the overall number of females becoming gravid in a population over time, not a reduction in clutch size per individual. While the mechanisms of action have yet to be defined, these results indicate that sublethal endosulfan concentrations may have a negative effect on grass shrimp reproductive biology.

Feasibility and safety of neural tissue transplantation in patients with syringomyelia.

Transplantation of fetal spinal cord (FSC) tissue has demonstrated significant potential in animal models for achieving partial anatomical and functional restoration following spinal cord injury (SCI). To determine whether this strategy can eventually be translated to humans with SCI, a pilot safety and feasibility study was initiated in patients with progressive posttraumatic syringomyelia (PPTS). A total of eight patients with PPTS have been enrolled to date, and this report presents findings for the first two patients through 18 months postoperative. The study design included detailed assessments of each subject at multiple pre- and postoperative time points. Outcome data were then compared with each subject's own baseline. The surgical protocol included detethering, cyst drainage, and implantation of 6-9-week postconception human FSC tissue. Immunosuppression with cyclosporine was initiated a few days prior to surgery and continued for 6 months postoperatively. Key outcome measures included: serial magnetic resonance imaging (MRI) exams, standardized measures of neurological impairment and functional disability, detailed pain assessment, and extensive neurophysiological testing. Through 18 months, the first two patients have been stable neurologically and the MRIs have shown evidence of solid tissue at the graft sites, without evidence of donor tissue overgrowth. Although it is still too soon to draw any firm conclusions, the findings from the initial two patients in this study suggest that intraspinal grafting of human FSC tissue is both feasible and safe.

Neurophysiological assessment of the feasibility and safety of neural tissue transplantation in patients with syringomyelia.

The feasibility and safety of a procedure involving fetal spinal cord tissue transplantation in patients with syringomyelia was assessed using a neurophysiological protocol designed to quantitate peripheral nerve function, spinal cord reflex excitability, and spinal cord conduction pathways essential for somatosensory evoked potentials. We report here data obtained before and for 18 months following the transplantation procedure performed on the first two patients in this study. The neurophysiological assessment protocols included measures of cortical and spinal cord evoked potentials, H-reflex excitability, and peripheral nerve conduction. Prior to the procedure, both patients had significant deficits on some of the neurophysiological measures, for example, lower extremity cortical evoked potentials. However, robust measures of intact pathways, such as upper extremity cortical evoked potentials, were also observed preoperatively in both patients. Thus, it was anticipated that conduction in these intact pathways could be at risk either from complications from the transplantation procedure and/or from continued expansion of the syrinx. Following the transplantation procedure, no negative changes were observed in any of the neurophysiological measures in either patient. In addition, patient 1 showed a decrease in the rate potentiation of tibial H-reflexes on the right side and an increase in the response probability of left tibial H-reflexes. The results of this postoperative longitudinal assessment provide a first-level demonstration of the safety of the intraspinal neural tissue transplantation procedure. However, the consideration of safety is currently limited to the grafting procedure itself, since the long-term fates of the donor tissue in these two patients remain to be shown more definitively.

Visualization of neural tissue water compartments using biexponential diffusion tensor MRI.

The apparent diffusion tensor (ADT) imaging method was extended to account for multiple diffusion components. A biexponential ADT imaging experiment was used to obtain separate images of rapidly and slowly diffusing water fractions in excised rat spinal cord. The fast and slow component tensors were compared and found to exhibit similar gross features, such as fractional anisotropy, in both white and gray matter. However, there were also some important differences, which are consistent with the different structures occupying intracellular and extracellular spaces. Evidence supporting the assignment of the two tensor components to extracellular and intracellular water fractions is provided by an NMR spectroscopic investigation of homogeneous samples of brain tissue. Magn Reson Med 45:580-587, 2001.

Determination of acute mortality in adults and sublethal embryo responses of Palaemonetes pugio to endosulfan and methoprene exposure.

Adult grass shrimp (Palaemonetes pugio) were exposed to endosulfan or methoprene for 96 h and LC(50) values were calculated. Male and female P. pugio cohorts were also exposed to endosulfan for 96 h in an attempt to determine potential differences in sensitivity between the sexes. Results from the methoprene exposure indicated that this pesticide was not acutely toxic to adult grass shrimp at 1 mg l(-1). Due to the lack of sensitivity, sex specific tests with methoprene were not performed. The calculated LC(50) for a population of grass shrimp, including both males and females exposed to endosulfan, was 0.62 microg l(-1). The LC(50) determinations for the sex specific tests were 0.92 microg l(-1) for males and 1.99 microg l(-1) for females. Following these acute exposures, reproductively active grass shrimp were chronically exposed to 200 ng l(-1) endosulfan or 1 mg l(-1) methoprene and were allowed to produce embryos. The resulting embryos were assessed for potential sublethal toxicity. There were no observed differences in the percent successfully hatching or larval mortality 3-days post hatch among the treatments. However, endosulfan treated embryos had a significantly increased hatching time (9.76 days compared to 8.72 days in controls). Methoprene treated embryos also took longer to hatch (9.55 days), but this delay was not significantly different from controls. These findings suggest that endosulfan may preferentially affect male grass shrimp and exposed female grass shrimp may produce embryos with delayed hatching times.