Effects of different incubation temperatures on the yolk‐feeding stage of Eupallasella percnurus(Pallas)

Embryos and yolk‐feeding larvae of lake minnow Eupallasella percnurus were reared at 13, 16, 19, 22 and 25° C with no access to external food. Time from egg activation to first embryonic movements, hatching, filling of swimbladder and final yolk resorption increased with decreasing temperature. At 13° C, c . 40% of larvae were unable to fill their swimbladder. The predicted lower temperature at which development and growth ceased (biological zero, t ₀) was the same for both processes, c . 7·5–10·5° C. There was no ontogenetic shift in the t ₀ value. Temperature coefficients for development ( Q _10dev.) ranged from 2 to 3 at 19–25° C, but were higher in hatched larvae at lower temperatures. Eggs of E. percnurus had a combination of small size, high hydration and low caloric value of fresh matter. Dry mass of larval tissue on yolk, percentage of dry matter in wet matter, and specific growth rate were maximized at 22 and 25° C. At 19–25° C, energy and matter contained in the initial eggs were converted to body tissue most efficiently. Temperatures from 22 to 25° C are considered optimal for E. percnurus embryos and yolk‐feeding larvae and are recommended for their indoor rearing.     

Effects of river flow on larval growth and survival of Japanese seaperch Lateolabrax japonicus (Pisces) in the Chikugo River estuary, upper Ariake Bay

The abundance and growth history of larval and juvenile Japanese seaperch Lateolabrax japonicus were investigated in the Chikugo River estuary, upper Ariake Bay, from 1990 to 2000. Growth during the larval period (up to 15 mm standard length, L _S, the size at recruitment into the estuary) was backcalculated using sagittal otolith microstructures by the biological intercept method. Growth rates in length declined at body sizes >14 mm L _S. High freshwater discharge through the Chikugo River was associated with high temperatures of the upper Ariake Bay where the larvae spend their planktonic life. Mean larval stage duration (days) from hatch to 15 mm ( D ₁₅) varied between 48·8 and 76·2 days and was inversely correlated with the estimated mean temperature history [mean daily temperature (° C) experienced by the larvae during the period from hatch to 15 mm, T ₁₅]. Mean abundance (number m⁻²) of larvae and juveniles was highest in years when T ₁₅, D ₁₅ and freshwater discharge were at intermediate levels. Although the abundance was not correlated with either of these variables, an exponential relationship between abundance and D ₁₅ was found when data collected during the highest river discharge years (1990, 1991 and 1998) were excluded. The increase in freshwater discharge through the Chikugo River probably had the potential to enhance or diminish Japanese seaperch recruitment in two ways: 1) it could increase recruitment probability by increasing temperature and larval growth and 2) high river flow also had the potential to decrease the probability of immigration into the river by increasing larval seaward dispersion, predation due to decreased turbidity and starvation due to decreased zooplankton prey abundance in the estuary.     

Effects of development, temperature and salinity on metabolism in eggs and yolk-sac larvae of milkfish, Chanos chanos (Forsskål)

Oxygen uptake rates and yolk-inclusive dry weiGhts were measured during the egg and yolk-sac larval stages of milkfish, Chanos chanos (Forsskal). Oxygen uptake by eggs and yolk-sac larvae was measured to assess the effects of four salinities (20,25,30,35 ppt) at 28°C. The effects of three temperatures (23,28,33°C) on oxygen uptake by yolk-sac larvae were determined at a salinity of 35 ppt. Dry weights were measured throughout embryonic development at 28°C and the yolk-sac stage at 23.28 and 33°C.
Oxygen uptake rates of eggs increased more than fivefold during embryogenesis (0.07±0.03 to 0.40 ± 03 μl O₂ egg ⁻¹ h ⁻¹;blastula to prehatch stage). Larval oxygen uptake did not change with age but was affected by rearing temperature (0.33 ± 0.08, 0.44 ± 0.07 and 0.63 ± 0.13 μl O₂ larva ⁻¹ h⁻¹ at 23, 28 and 33°C, respectively; Q₁₀= 1.93). Acute temperature changes from 28 to 33°C caused significant increases in oxygen uptake by embryos (Q ₁₀= 1.69–3.58) and yolk-sac larvae (Q ₁₀=2.55). Salinity did not affect metabolic rates.
Dry weight of eggs incubated at 28°C decreased 13% from fertilization to hatching. Incubation temperatures from 23–33°C did not affect dry weights at hatching. Rearing temperatures significantly affected the rate of larval yolk absorption (Q ₁₀= 2.25).     

Effects of river flows on growth of redbreast sunfish Lepomis auritus (Centrarchidae) in Georgia rivers

Effects of river discharge on growth of redbreast sunfish Lepomis auritus were investigated in nine rivers in Georgia, U.S.A. Fish were aged and annular total length increments ( L _Tinc) estimated from measurements from sectioned sagittal otoliths using the generalized regression model that held for the effects of decreasing L _Tinc from annual age ( X ): L _Tinc= b _o− b ₁( X ) ± b _i( D ), where b _o, b ₁ and b _i were the regression coefficients for the intercept and slopes and D , discharge, was either a single or multiple measurements of annular or seasonal flow volume or variation in flow volume. For eight of nine rivers, higher or greater variation in flows from April to June was associated with greater L. auritus growth; in the last river, higher flows from January to March were associated with greater fish growth. Across all rivers, L. auritus growth increments were 22, 45 and 36% greater in a wet year v . a dry year at ages 1, 2 and 3 years, respectively. Based on the results of this study, increasing water withdrawals by an additional 30% in five Georgia rivers would reduce the predicted number of L. auritus recruiting to 203 mm (angler preferred size) by 19–62%.     

Non-destructive optical detection of pigment changes during leaf senescence and fruit ripening

Reflectance spectra in the visible and near infra-red range of the spectrum, acquired for maple ( Acer platanoides L.), chestnut ( Aesculus hippocastanum L.), potato ( Solanum tuberosum L.), coleus ( Coleus blumei Benth.), leaves and lemon (Citrus limon L.) and apple ( Malus domestica Borkh.) fruits were studied. An increase of reflectance between 550 and 740 nm accompanied senescence-induced degradation of chlorophyll (Chl), whereas in the range 400–500 nm it remained low, due to retention of carotenoids (Car). It was found that both leaf senescence and fruit ripening affect the difference between reflectance ( R ) near 670 and 500 nm ( R ₆₇₈− R ₅₀₀), depending on pigment composition. The plant senescing reflectance index in the form ( R ₆₇₈− R ₅₀₀)/ R ₇₅₀ was found to be sensitive to the Car/Chl ratio, and was used as a quantitative measure of leaf senescence and fruit ripening. The changes in the index were followed during leaf senescence, and natural and ethylene-induced fruit ripening. This novel index can be used for estimating the onset, the stage, relative rates and kinetics of senescence/ripening processes.     

Seasonal variability in growth of larval Japanese anchovy Engraulis japonicus driven by fluctuations in sea temperature in the Kii Channel, Japan

Seasonal variability in the growth of larval Japanese anchovy Engraulis japonicus was examined through otolith microstructure analysis based on the samples collected from the northern side (inner area, IA) and the southern side (outer area, OA) of the Kii Channel from April 2006 to March 2007. Growth trajectories (otolith backcalculated mean standard length of 5 day intervals from 5 days after hatch to 24 days) as well as the most recent 5 day mean growth rate of larvae before capture ( G ₅) differed among months. Growth trajectories showed the same pattern as G ₅. In IA, mean ± s.d. G ₅ ranged from 0·31 ± 0·04 mm day⁻¹ (January) to 0·73 ± 0·06 mm day⁻¹ (October). In OA, mean ± s.d. G ₅ ranged from 0·36 ± 0·05 mm day⁻¹ (January) to 0·79 ± 0·11 mm day⁻¹ (August). G ₅ values declined from November to January and then started to increase. In general, the seasonal patterns of growth were similar between IA and OA, and a clear seasonal pattern in growth was identified. When the relationships among larval growth rate, sea temperature, zooplankton density and larval density were examined, growth rate was positively related with sea temperature in both areas and not related with the other factors. The similar pattern in growth observed between IA and OA was probably due to the low spatial variability in sea temperature compared to its seasonal variability.     

Oxygen uptake in developing eggs and larvae of the cod, Gadus morhua L.

Unfertilised cod eggs showed a mean oxygen uptake rate at 5°C of 0.089 μl O₂, dry wt.⁻¹ h⁻¹; this gradually rose to 0.768 μl O₂ mg dry wt.⁻¹ h⁻¹ in eggs about to hatch. From hatching to complete yolk absorption larvae respired at 1.6 μl O₂, mg dry wt.⁻¹ h⁻¹. During starvation following yolk absorption, uptake fell significantly to 1.1 μl O₂, mg dry ⁻¹ h⁻¹. Much of this decrease in oxygen consumption was shown to be caused by reduction in activity. Loss of weight during the embryo and larval phases could not easily be reconciled with total oxygen consumption; it is suggested that cod embryos and larvae may not rely solely upon endogenous energy reserves during development.     

Gastric evacuation in horse mackerel. I. The effects of meal size, temperature and predator weight

Gastric evacuation experiments were performed on horse mackerel Trachurus trachurus. A nearly full matrix experimental design with respect to the variables predator weight (<10–400 g) meal size (up to 7·8% body weight) and temperature (10–20°) was covered with 0-group smelt Osmerus eperlanus as prey. A general evacuation model without meal size as a variable was fitted to the data on wet weights as well as on dry weights by means of non-linear regression technique. Two methods of data transformation, relative data and square root transformation, were applied to improve variance homogeneity. The most reliable model fit was achieved on dry weight data applying the square root transformation technique: where S_t=stomach content (g wet weight) at time t after ingestion, S₀=the initial meal size, W =predator (g wet weight), and T =temperature. The estimated coefficient of the exponential temperature function, δ=00·032, corresponds to a Q ₁₀ value of 1·4 which is outstandingly low in comparison with results on other species. However additional experiments to determine maximum daily food rations indicated that appetite in contrast to gastric evacuation is strongly temperature dependent.     

Mechanism of docosahexaenoic acid-induced inhibition of in vitro Aβ1–42 fibrillation and Aβ1–42-induced toxicity in SH-S5Y5 cells

The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n -3), one of the essential brain nutrients, on in vitro fibrillation of amyloid β (Aβ_1–42), Aβ_1–42-oligomers and its toxicity imparted to SH-S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Aβ_1–42-fibrill formation with a concomitant reduction in the levels of soluble Aβ_1–42 oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%), Tris–Tricine gradient(4–20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Aβ_1–42 oligomers of 15–20 kDa, indicating that it hinders these on-pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Aβ_1–42 backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern–Volmer constants. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction efficiency and immunohistochemical examination suggested that DHA inhibits Aβ_1–42-induced toxicity in SH-S5Y5 cells. Taken together, these data suggest that by restraining Aβ_1–42 toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer's disease.     

Physical factors affecting the toxicity of sprays to stored product insects; the quantity of carrier in which a given amount of active ingredient is applied

The problem of whether a given amount of active ingredient is more effectively applied in concentrated or dilute form is discussed. If y is percentage mortality and x ₁ and x ₂ are respectively log-concentration of active ingredient and log-volume (or deposit) of insecticide applied, y may be regarded as a uniform, continuous function of x ₁ and x ₂. The amount of active ingredient is constant, i.e. x ₁+ x ₂= k , so that
dx/dx₁=−dy/dx₂=δy/δx₁−δy/δx₂
Probit mortality, Y , can be substituted for y in (4). Thus, whether an active ingredient is better applied in concentrated or dilute form depends on the relative magnitudes of ∂ y /∂ x ₁ and ∂ y /∂ x ₂, or of ∂ Y /∂ x ₁ and ∂ Y /∂ x ₂. Equation (4) is true whenever an insecticide consists of an active ingredient in a diluent, whatever the dosage-mortality relationship. Previous work is discussed in the light of (4) and its probit form, and it appears that the concentration at which an active ingredient is best applied can depend upon the nature and quantity of the active ingredient, and the method of application of the spray. There may be other facto*** The probit form of (4) is applied to the probit plane and confirmed experimentally. Flour beetles, Tribolium castaneum Herbst were sprayed with pyrethrins in Shell oil P 31, and it was found that ∂ Y /∂ x ₁ > ∂ Y /∂ x ₂, so that a given quantity of pyrethrins was more toxic in concentrated solution than in dilute.     

Mortality and production of 0+ perch, Perca fluviatilis L., in two Scottish lakes

Mortality of 0+ Eurasian perch was measured directly from the decline in catch in a high-speed plankton sampler and described by a negative exponential model. A mean mortality of 0.063 day ¹ (range=0.020–0.089) was calculated; for 0+ juveniles it was 0.021 day ¹. Mean larval production was 0.85–1.30 mg dry weight m⁻² day⁻¹ in Loch Kinord and 0.08–0.12 in Loch Davan. Annual production of 0+ juveniles was 1.97–7.55 kg wet weight ha ⁻¹ at L. Kinord and 1.01 kg at L. Davan. The contribution of 0+ perch, including the egg stages, to total perch production was 88–96%. Availability of food items on transition to exogenous feeding and through the larval period did not limit survival. Cannibalism by adults accounted for most of larval mortality, and predation by pike and adult perch were responsible for the majority of juvenile losses. The population structure of adult perch, for 12 year classes, was stable at L. Kinord, while year classes at L. Davan exhibited fluctuations in abundance and in two sampling years were eliminated in the first summer. Survival in the eleutheroembryonic phase was variable at L. Davan and observed to be low compared with L. Kinord. It is suggested that this was a result of water turbulence on the exposed and shallow spawning areas at L. Davan.     

Effects of egg composition and prey density on the larval growth and survival of lake whitefish (Coregonus clupeaformis Mitchill)

Eggs were collected from two stocks of lake whitefish, Coregonus clupeaformis , in Lakes Michigan and Huron to assess the effect of egg composition and prey density on larval growth and survival. Egg composition parameters including wet weight (mg egg⁻¹), dry weight (mg egg⁻¹), percent water, total caloric content (cal egg⁻¹), caloric density (cal egg⁻¹), percent lipid content, and total lipidcontent (mg egg⁻¹) were measured. Fish hatched from six parental females in each stock were fed one of four rations (0, 18, 24, 50 brine shrimp larva⁻¹ day⁻¹) after yolk sac absorption. Length at hatch, endogenous growth, exogenous growth, and survival were measured during a 42-day laboratory experiment. Length at hatch of larvae was positively related to egg caloric content ( r ²=0.780). Endogenous growth for lake whitefish larvae was positively related to percent lipid content ( r ²=0.896) and total egg lipid content ( r ²=0.876) of parental females. Exogenous growth and survival of larval lake whitefish was positively related to prey availability. Larval fish growth was accurately modelled ( r ²=0.973) as a function of prey abundance using a threshold-corrected hyperbolic equation. These results indicate that both egg composition and prey availability have the potential to influence the growth and survival dynamics of larval lake whitefish significantly.     

Age, growth and mortality of the Mediterranean amberjack Seriola dumerili (Risso 1810) from the south-eastern Adriatic Sea

The age, growth and mortality of the Mediterranean amberjack Seriola dumerili (Risso 1810) were determined in 298 specimens collected in the south-eastern Adriatic Sea (Donji Molunat Bay) from 17 May to 26 June 1997. The total length ranged from 32.0 to 160.0 cm and weight from 0.5 to 46.5 kg. Ten age classes, ranging from 1° to 10° years were defined by scale readings. Mean total length and weight-at-age data were used to estimate the growth parameters of the von Bertalanffy equation: L _∞=174.6 cm, K =0.190, t ₀=–0.314; W _∞=79.01 kg, K =0.139 and t ₀=–0.746. The length–weight relationship was estimated at: W =0.000123 ×  L ^2.847. The overall sex ratio was 1 : 1.06 in favour of males. Total ( Z ) and natural ( M ) mortality were found to be 0.41 years⁻¹ and 0.30 years^–1, respectively. The exploitation ratio ( E =0.27) indicates that the fishing pressure on the Mediterranean amberjack was low in the investigated region.     

Early development of milkfish: effects of salinity on embryonic and larval metabolism, yolk absorption and growth

During embryogenesis of Chanos chanos , more than half of the yolk was consumed and the majority of it was converted into larval tissue. Salinity affected both yolk absorption and embryonic and larval growth. Larvae hatched in 20% had larger yolk reserves but were smaller and grew more slowly than larvae in 35 and 50%. Larvae hatched in 35 and 50% had equal amounts of yolk but those from 35% were larger. Oxygen consumption rates increased during development (from 0.06 ± 0.01 μl O₂ egg^–1 h^–1 by blastulae to 0.37 ± 0-01 μl O₂ egg^–1 h^–1 by prehatch embryos and 0–43 ± 0–03 μl O₂ larva ^–1 h ^–1 by newly-hatched larvae) and were significantly affected by salinity. Eggs and yolk-sac larvae incubated in 35% consumed more oxygen than those in the low and high salinities. Salinity affected both the rate and pattern of yolk utilization but salinity-related differences in metabolism, yolk absorption, and growth were not related directly to the osmotic gradient. Low salinity retarded yolk absorption while high salinity reduced yolk utilization efficiencies. Differences in oxygen consumption rates were probably related to variations in the relative amounts of metabolically active embryonic and larval tissue and/or higher activity levels rather than differential osmoregulatory costs. 35% is probably the most suitable salinity for incubation and larval rearing of milkfish.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

Duration of synchronous cleavage cycles and rate of development at different temperatures in the Baltic herring

The duration of one synchronous cleavage cycle (τ₀) in Clupea harengus membras at different temperatures ( T ) was given by: (logτ₀)= 2.4349–0–0684T for T= 0.9–13°C, and (logτ₀)= 1.61010–oooit for t= 13–18–7°C.     

Seasonal variations in the energy density of fishes in the North Sea

The energy density ( E _D, kJ g^-1 wet mass) of saithe Pollachius virens , haddock Melanogrammus aeglefinus , whiting Merlangius merlangus , Norway pout Trisopterus esmarki , herring Clupea harengus , sprat Sprattus sprattus , sandeel Ammodytes marinus and pearlsides Maurolicus Muelleri , from the North Sea, increased with total length, L _T. However, there was not always a significant ( P> 0·05) linear relationship between L _T and E _D. Seasonal differences in E _D were obvious in mature fish, while geographical differences were insignificant. For all species there was a highly significant correlation ( P< 0·0001) between the percent dry mass of the fish ( D S) and E _D. A general relationship was established for gadoids and sandeel E _D=–3·1492+0·3459 D _S and herring E _D=–4·6395+0·4170 D _S. Thus seasonal and size-specific data on E _D needed for bioenergetics and gastric evacuation models can be determined simply from D _S, which is considerably less costly and time consuming than calorimetry or proximate analysis.     

Isoform-Specific Effects of Apolipoproteins E2, E3, and E4 on Cerebral Capillary Sequestration and Blood-Brain Barrier Transport of Circulating Alzheimer's Amyloid β

Abstract: Cerebral capillary sequestration and blood-brain barrier (BBB) permeability to apolipoproteins E2 (apoE2), E3 (apoE3), and E4 (apoE4) and to their complexes with sAβ_1–40, a peptide homologous to the major form of soluble Alzheimer's amyloid β, were studied in perfused guinea pig brain. Cerebrovascular uptake of three apoE isoforms was low, their blood-to-brain transport undetectable, but uptake by the choroid plexus significant. Binding of all three isoforms to sAβ_1–40 in vitro was similar with a K _D between 11.8 and 12.9 n M . Transport into brain parenchyma and sequestration by BBB and choroid plexus were negligible for sAβ_1–40-apoE2 and sAβ_1–40-apoE3, but significant for sAβ_1–40-apoE4. After 10 min, 85% of sAβ_1–40-apoE4 taken up at the BBB remained as intact complex, whereas free sAβ_1–40 was 51% degraded. Circulating apoE isoforms have contrasting effects on cerebral capillary uptake of and BBB permeability of sAβ. ApoE2 and apoE3 completely prevent cerebral capillary sequestration and blood-to-brain transport of sAβ_1–40. Conversely, apoE4, by entering brain microvessels and parenchyma as a stable complex with sAβ, reduces peptide degradation and may predispose to cerebrovascular and possibly enhance parenchymal amyloid formation under pathological conditions.     

Growth, diet and metabolism of common wolf–fish in the North Sea, a fast–growing population

The von Bertalanffy growth parameters for common wolf–fish Anarhichas lupus in the North Sea were: male: L ∞=111·2 cm, t ₀=–0·43 and K =0·12; and female: L ∞=115·1 cm, t ₀=–0·39 and K =0·11, making this the fastest growing stock reported. Resting metabolic rates (RMR±S.E.) and maximum metabolic rates (MMR±S.E.) for six adult common wolf–fish (mean weight, 1·39 kg) at 5° C were 12·18±1·6 mg O₂ kg^–1 h^–1 and 70·65±7·63 mg O₂ kg^–1 h^–1 respectively, and at 10° C were 25·43±1·31 mg O₂ kg^–1 h^–1 and 113·84±16·26 mg O₂ kg^–1 h^–1. Absolute metabolic scope was 53% greater at 10° C than at 5° C. The diet was dominated by Decapoda (39% overall by relative occurrence), Bivalvia (20%) and Gastropoda (12%). Sea urchins, typically of low energy value, occupied only 7% of the diet. The fast growth probably resulted from summer temperatures approximating to the optimum for food processing and growth, but may have been influenced by diet, and reduced competition following high fishing intensity.     

Effects of copper, lead and zinc in soil on egg development and hatching of Folsomia candida

Effects of CaCl₂, CuCl₂, ZnCl₂ and PbCl₂ on development and hatching success of eggs of Folsomia candida (Collembola) were studied under laboratory conditions. Thousands of healthy eggs from synchronized cultures were incubated in soils treated with different concentrations of the metals. Compared with the water control, egg hatch significantly decreased when concentrations of Cu, Pb and Zn reached 400, 1600 and 800 mg/kg dry soil, respectively. Values of EC_{50 (hatching)}, calculated according to the exponential model (with 95% confidence limits in brackets), were 625 (407–875), 2361 (2064–2687) and 1763 (1548–2000) mg/kg dry soils for Cu, Pb and Zn, respectively. When Cu concentration reached 1600 mg/kg dry soil, eggs became green and the percentage of green eggs changed from 5%–20% after incubation for 2 days to 15%–30% after incubation for 4 days. At 3200 mg Cu/kg dry soil, tissues inside eggs were black and shrunken.