Impact of 17alpha-ethinylestradiol on the plankton in freshwater microcosms--II: responses of phytoplankton and the interrelation within the ecosystem

Effects of 17α-ethinylestradiol (EE) on phytoplankton were investigated in a lentic freshwater microcosm study. Treatment with EE caused a shift in the species composition as shown by a principle response curve. Whereas densities of Cyanophyceae, Dinophyceae, and Chrysophyceae increased, those of Conjugatophyceae and Cryptophyceae decreased. Furthermore, relative density of Chlorophyceae declined after EE treatment. The changes showed taxa-specific time dependencies. Some species, especially the cyanobacterium Cyanobium parvum, bloomed after the treatment. EE treatment promoted total abundance and biomass of phytoplankton. Although the number of species per microcosm increased, the diversity indices (Shannon–Wiener, Simpson) tended to lower values. The ecosystem only partly recovered during the investigated post-treatment period of 6 weeks. Probably at least the main effects were caused indirectly, i.e. via decrease of grazing zooplankton (crustaceans). The relation of EE to variation of phytoplankton composition was closer than those of other abiotic factors, indicating the relevance of its impact. EE also probably affected nutrients of the phytoplankton.     

Influence of 4-nonylphenol on the structure of nematode communities in freshwater microcosms

We investigated the effect of 4-nonylphenol (NP) on nematode communities in the sediment of freshwater microcosms. Seven treatments were dosed with various concentrations of NP over a period of six weeks by using a controlled-release method (NPI-NP7; maximum sediment concentrations: 0.29-3.37 mg/kg dry wt). Four microcosms were not exposed to NP and served as controls. Nematode communities were analyzed over a period of 15 weeks, including sampling dates before, within, and after the NP application. Communities were characterized in terms of total nematode abundance and species diversity (Shannon index and evenness), as well as composition of species, feeding types, and different life-history strategists (maturity index [MI]). Species composition was analyzed by using a multivariate method (principal response curves). Total nematode abundance and species diversity were not affected in any of the NP-treated microcosms. However, in the highest dosed treatment, NP-induced changes in the nematode communities occurred. Species and feeding types composition, as well as the MI, were affected in the postapplication period, with species composition being altered most clearly. In the highest dosed treatment, deposit-feeding species, classified as colonizers (Eumonhystera), increased in dominance, whereas epistrate feeders and chewers (Prodesmodora and Tobrilus) decreased in relative abundance compared to the control.     

Effects of nonylphenol on zooplankton in aquatic microcosms

We investigated the effects of the endocrine disruptor nonylphenol (NP) on the zooplankton assemblages of 230 L aquatic microcosms during a four-week preapplication period, a six-week NP treatment via controlled release, and a six-week postapplication period. Zooplankton assemblage change, investigated by ordination principal response curves (PRC), was due to lower abundances of copepoda, rotifera, and cladocera. The most sensitive groups/taxa were copepoda larvae, followed by the rotifers Synchaeta spp., Polyarthra spp., and the cladocerans Daphnia longispina and Chydorus sphaericus. The mean no-observed-effect concentrations for the community (NOEC(community)) was 30 microg/L. Cladocera densities recovered during the postapplication period at all but the highest NP concentrations (maximum 120 microg/L); copepod densities did not recover at the three highest concentrations (maximum 96-120 microg/L).     

Effect of extremely severe winters on under-ice phytoplankton development in a mesotrophic lake (Eastern Poland)

The research was carried out in a mesotrophic and dimictic lake during winters with ice cover. In the last forty years, the development of phytoplankton was analyzed in five extreme winter seasons. The studies of phytoplankton characteristics in the water column took into account values of biomass, concentration of chlorophyll-a and species composition, including dominant species. Differences in the vertical distribution of flagellate and non-flagellate species belonging to cyanobacteria and algae were analyzed in the gradient of light and thermal conditions. The phytoplankton biomass was low and vertically differentiated, with the lowest values at the deeper part of the water column. Flagellate species from the group of Cryptophyceae, Chrysophyceae and Dinophyceae were most abundant. Species biodiversity was low but every winter the dominant species represented different taxonomic groups. In some periods, larger non-motile phytoplankton species from green or blue-green algae dominated. The research proved that the development of phytoplankton under the ice cover was limited mainly by light and, to a lesser extent, by temperature.     

Atrazine does not affect algal biomass or snail populations in microcosm communities at environmentally relevant concentrations

The herbicide atrazine is a photosynthetic inhibitor used around the world in agricultural applications. Contamination of surface waters adjacent to treated areas can directly reduce growth of nontarget aquatic autotrophs, but the severity of impacts is highly dependent on species sensitivity and exposure concentration. Secondary effects resulting from macrophyte or phytoplankton decline may include an expansion of the more tolerant periphyton community. Recently, this shift in the autotrophic community has been proposed as a mechanism for increased rates of parasite infections in amphibians via augmented populations of aquatic snails which act as intermediate hosts to larval trematodes. To further clarify this relationship, an outdoor microcosm study was conducted to examine the effects of atrazine on primary production and snail populations over a range of environmentally relevant concentrations. In July 2009, 15 experimental ponds were treated to achieve initial concentrations of 0, 1, 10, 30, and 100 μg/L atrazine. Over a period of 73 d, measures were taken of macrophyte, phytoplankton, and periphyton biomass, growth, and fecundity of caged snails (Physella spp. and Stagnicola elodes) and free-living snails (Physella spp.). Except for declines in macrophyte biomass at the highest treatment level, no consistent relationships were found between atrazine concentration and any measured parameter. Comparison of these results with previous findings highlights the variability of responses to atrazine exposure between similarly constructed freshwater communities, even at concentrations up to 20 times higher than sustained environmental levels.     

Effects of pulp mill effluent on benthic assemblages in mesocosms along the Saint John River, Canada

We used mesocosms to examine the impact of different concentrations of pulp mill effluent (PME) on structural and functional endpoints of a benthic assemblage in the Saint John River (NB, Canada) during 1999 and 2000. Previous studies on this effluent's effects produced conflicting results, with field surveys suggesting a pattern of mild nutrient enrichment, while laboratory toxicity tests linked effluent exposure to moderate contaminant effects. Experimental treatments included three concentrations of sulfite pulp mill effluent (0, 5, 10% v/v PME). Endpoints for the assessment included algal biomass and taxonomic composition, benthic invertebrate abundance and composition, and insect emergence. Low concentrations of PME increased periphyton biomass and caused changes in community structure within the diatom-dominated community. Pulp mill effluent addition had little effect on several structural endpoints measured for benthic invertebrates, including abundance and taxonomic richness, but significantly changed community composition. For both periphyton and benthic invertebrates, community composition endpoints were more sensitive indicators of PME exposure. Insect emergence was a highly relevant functional endpoint. When benthic and emerged insects were combined, total abundance increased with PME addition. Results from two trophic levels, which provided multiple lines of evidence, indicated that the main impact of these PME concentrations is nutrient enrichment rather than effluent toxicity. Our findings also suggest that benthic invertebrate and periphyton assemblages, algal biomass production, and insect emergence are sensitive response measures. Future studies may confirm this observation. The consideration of both functional and structural endpoints at different trophic levels can greatly improve our understanding the effects of discharges to rivers. Such an understanding could not have been obtained using standard assessment techniques and illustrates the value of mesocosms and the benthic community assemblage approach in environmental assessment.     

Impact of 17alpha-ethinylestradiol on the plankton in freshwater microcosms--I: response of zooplankton and abiotic variables

We investigated effects of 17alpha-ethinylestradiol (EE) in vertebrate free 230 L still water microcosms. Zooplankton composition and physico-chemical variables were observed during 4 weeks of pre-application, 6 weeks of dosing via controlled release, and a 12 weeks post-treatment period. In the treated microcosms, time-weighted averages of EE concentration ranged between 7 and 220 ng/L during the dosing period, with concentration maxima up to 724 ng/L. EE exposure resulted in a decrease of species numbers and diversity (Shannon-Wiener, Simpson). Abundances of cladocerans, copepods, and, less unambiguously, rotifers declined. Strongest affected groups were the offspring of cladocerans and copepods and, on species level, the cladoceran species Daphnia longispina and Chydorus sphaericus as well as the rotifer species Keratella quadrata and Polyarthra sp. EE apparently affected the phosphate cycle as indicated by increased phosphate concentrations in the water. During post-treatment period, the treated microcosms recovered, but especially the highest treated microcosms did not fully re-approximate to the controls. Whereas EE affected cladocerans and copepods directly, shifts of rotifers may (partly) be caused indirectly, e.g. by competition with crustaceae. Although not providing an absolute proof, the traits of direct and indirect effects on different taxonomic groups and larval stages as well as the time course of the effects indicate that effects primarily resulted from endocrine activity of EE.     

Effect of a copper gradient on plant community structure

Vegetation data including plant cover, biomass, species richness, and vegetation height was sampled on a copper-contaminated field with total copper contents varying from 50 to almost 3,000 mg/kg soil. The field was covered by early succession grassland dominated by Agrostis stolonifera. Plant cover, biomass, species richness, and vegetation height generally decreased with increasing copper content, although the highest biomass was reached at intermediate copper concentrations. Multivariate statistical analyses showed that plant community composition was significantly correlated with soil copper concentration and that community composition at soil copper concentrations above 200 mg/kg differed significantly from community composition at lower copper levels. Comparison of single-species (Black Bindweed, Fallopia convolvulus) performance at the field site and in laboratory tests involving field soil and spiked soil indicates that the laboratory tests conventionally applied for risk assessment purposes do not overestimate copper effects. Interaction between copper and other stressors operating only in the field probably balance the higher bioavailability in spiked soil.     

Effects of fomesafen, alone and in combination with an adjuvant, on plankton communities in freshwater outdoor pond mesocosms

Ecotoxicological effects of the diphenyl ether herbicide fomesafen, applied alone or in combination with the adjuvant Agral 90 (mixture of polyethoxylated derivatives of nonylphenol), were assessed on planktonic communities in 18-m3 outdoor mesocosms during a nine-month study. Four mesocosms were treated with fomesafen only (nominal concentration: 40 microg/L), four were treated with the mixture fomesafen-Agral 90 (nominal concentration: 40 microg/L and 90 microg/L, respectively), and four were kept as the controls. Five treatments were performed every three weeks from April 18, 2000. Mean (+/- standard error [SE]) values of fomesafen concentration in water of 62.5 (+/-5.3) and 19.4 (+/-7.6) microg/L were measured at the end of the treatment period in fomesafen- and mixture-treated mesocosms, respectively. Fomesafen, either alone or in mixture with Agral 90, had a significant positive effect on the abundance and biovolume of Cyanobacteria, Cryptophyceae, Dinophyceae, and Bacillariophyceae. Chlorophyceae were inhibited by the herbicide and laboratory toxicity tests confirmed that green algae were more sensitive toward fomesafen than other algal classes. A positive effect of treatments on phytoplankton taxonomic diversity also was observed, indicating that, like natural disturbances of intermediate strength, xenobiotics sometimes may enhance the diversity of algal communities. Fomesafen alone did not have any clear effect on zooplankton. Abundance of calanoid copepods was reduced significantly in the mixture-treated ponds, suggesting either a direct effect of the adjuvant and/or an enhancement of herbicide toxicity by Agral 90. The abundance of other zooplanktonic herbivorous groups increased due to a reduced competition for food for herbivorous species and a higher availability of preys for predators. No algal bloom was observed in the treated ponds, presumably because of grazing pressure and the low availability of nutrients.     

Response of Phytoplankton Community to Low-Dose Atrazine Exposure Combined with Phosphorus Fluctuations

The effects of atrazine on a controlled phytoplankton community derived from a natural freshwater wetland exposed to low doses of this photosynthesis-inhibiting herbicide were examined. The community was exposed for 7 weeks to doses of 0.1, 1, and 10 μg L⁻¹ atrazine, combined with changes in nutrient concentration, and the photosynthetic activity, biomass, and community structure were noted during the experiment. Responses of the phytoplankton community were examined in terms of photosynthetic activity, biomass, and community structure. Significant effects of atrazine on the phytoplankton assemblage, in terms of primary production and community structure, were highlighted, even at doses as low as 1 and 0.1 μg L⁻¹, when associated with phosphorus fluctuations. The most abundant Chlorophyceae decreased in concentration with increasing atrazine dose, whereas cyanobacteria were more tolerant to atrazine, particularly with increased nutrient supply. The subinhibitory doses of atrazine used in the present study confirmed the higher sensitivity of long-term exposure of multispecies assemblages under resource competition. Our study supports the emerging hypothesis that the increasing prevalence of cyanobacterial blooms in European aquatic systems may result from a combination of unbalanced nutrient enrichment and selective pressures from multiple toxicants.     

Phytoplankton of the Irkutsk Reservoir as an indicator of water quality

The species composition of phytoplankton and quantitative parameters of its abundance are indicators of ecological conditions in a water body. This paper deals with the analysis of phytoplankton in the Irkutsk Reservoir, which was formed more than 50 years ago on the Angara River flowing out of Lake Baikal. The level of phytoplankton development in the reservoir is directly dependent on the abundance of diatoms in Lake Baikal. The dominant assemblage of algal species is stable and consists mainly of diatoms, chlorophytes, and chrysophytes. Cyanobacteria are represented by a small number of species with low abundance. According to the composition of indicator species, the water in the lower reaches of the Irkutsk Reservoir can be classified as clean. The saprobity index in the open-water period is 1.2, increasing to between 1.3 and 1.5 in small bays. The values of phytoplankton biomass and chemical parameters of water recorded in 2008 indicate that the Irkutsk Reservoir is an oligotrophic water body. Its trophic status has not changed significantly since the time of impoundment (1956?C1958).     

Effects of Exposure Duration of Herbicides on Natural Stream Periphyton Communities and Recovery

To improve risk estimates for herbicides in streams, the sensitivity of natural periphyton communities to four herbicides (metribuzin, hexazinone, isoproturon, and pendimethalin) was examined in experiments including varying exposure duration and a recovery phase. Effect parameters included assimilation of ¹⁴C and concentration of diagnostic pigments as proxies for photosynthetic activity and algal group composition, respectively. The results indicated that isoproturon, metribuzin, and hexazinone affected the photosynthetic activity of periphyton at distinctly lower concentrations than the effect concentrations published for standard single-species growth-tests with phytoplankton species. Pendimethalin did not show effects on the photosynthetic activity of periphyton at the concentrations tested. The effect concentration (EC₅₀) of isoproturon and metribuzin decreased by one to two orders of magnitude when the duration of exposure increased from 1 h to 24 h, while hexazinone had a stimulating effect on the photosynthetic activity of periphyton after 1 h exposure and inhibiting effect after 24 h exposure. The photosynthetic activity after exposure to metribuzin for 1, 2, 6, 18, 23, or 48 h recovered almost completely after 48 h in herbicide-free water. However, different periphyton groups responded differently to metribuzin exposure: Chlorophytes were severely affected by exposure and did not recover, whereas diatoms and especially cyanobacteria recovered well. Overall the study showed that the effects of herbicides on periphyton are strongly affected by the duration of exposure, and even short-term exposure may have distinct effects on the periphyton community. Received: 11 April 2002/Accepted: 22 January 2003     

Temporal pattern prevails over spatial variability in phytoplankton communities from a subtropical water supply reservoir

Phytoplankton species are dominant components in reservoir ecosystems, yet little is known about their variability and dynamics, especially along the depth continuum. This study examined vertical and horizontal differences in phytoplankton communities in a typical subtropical deep reservoir (i.e. Dongzhen Reservoir) from 2011 to 2012. Phytoplankton communities separated into four groups based on the biomass data, indicating that temporal differences in the community structure were greater than spatial differences. Autumn communities had the highest diversity measured by the Shannon-Wiener index (2.47±0.07), while summer communities had the lowest diversity (0.46±0.09). Both winter and spring communities were dominated by diatoms (90.4±1.7%), while cyanobacteria dominated in summer communities during our sampling period. In spring and summer, however, the three surface communities characterized by high biomass were most similar to each other, indicating that vertical variation was significantly higher than horizontal differences. Furthermore, Cylindrospermopsis raciborskii accounted for over 96% of the total phytoplankton biomass in the summer surface water. The redundancy analysis (RDA) illustrated that the temporal factor (summer), the spatial factor (depth), and nutrients (nitrite and nitrate nitrogen, ammonium nitrogen, phosphate phosphorus) were significant variables affecting the dynamics of phytoplankton communities. High temperature with stable thermal stratification might have been the cause of C. raciborskii dominance in Dongzhen Reservoir in summer. Regular and long-term monitoring of dominant species is urgently needed for water quality protection and sustainable reservoir management. Copyright© of Dept. of Oceanography and Geography, University of Gdańsk, Poland     

Genetic changes in phytoplankton communities exposed to the herbicide simetryn in outdoor experimental ponds

Possible changes in the species composition of phytoplankton communities and the susceptibility of individual strains to the triazine herbicide, simetryn, were investigated in outdoor experimental ponds exposed to the herbicide. Centric diatoms remained predominantly longer in the treated ponds than in the control ponds during the early experimental period, but there was little difference in the overall floristic composition. In one of the two treatments, the phytoplankton community whose abundance had been suppressed by the chemical, subsequently became more abundant in spite of the much higher concentration of simetryn than previously. All the strains isolated from the herbicide-treated ponds during the latter experimental period were tolerant to 100 g/L simetryn (relative yields: >70%), whereas all the strains isolated from the control ponds at the same time were susceptible (relative yields: <15%). Scenedesmus gutwinskii var. heterospina showed a marked difference in susceptibility between the strains isolated from the treated and the control ponds. Tolerance of the isolated strains remained for nearly two years in the absence of simetryn, which indicate that changes in genetic composition occurred in the phytoplankton communities when they were exposed to the herbicide.     

Fate and shortterm persistence of the insecticide aminocarb in a New Brunswick (Canada) headwater stream

Aminocarb applied to the surface of a stream dispersed throughout the water column within 100 m of the place of application. Concentrations in the water were first highest near the surface, later more or less evenly distributed, and lingered longest near and within the stream bottom. Both oiland water-based formulations of Matacil^® 1.8F gave similar results. Aminocarb concentrations diminished logarithmically downstream.Concentrations of aminocarb in suspended particles, sediments, periphyton, higher plants, insects, and brook trout were not higher than ambient in water and dissipated quickly.Aminocarb phenol was found consistently in measurable concentrations in water treated with the aqueous formulation, but in much smaller concentrations in water treated with the oil formulation. It was also found only as very small amounts in three sediment samples in the stretch treated with the aqueous formulation.A slight increase in drift of Chironomidae was probably caused by aminocarb. No effects on survival, drift, or species composition of phytoplankton were noted.     

Ecological effects of an anionic C12-15 AE-3S alkylethoxysulfate surfactant in outdoor stream mesocosms

The ecological assessment of a C12-15 AE-3S linear alkylethoxysulfate (AES) anionic surfactant to invertebrates, fish, periphyton, and an aquatic macrophyte was conducted in a 30-d outdoor stream mesocosm study with five replicated concentrations and controls. Alkylethoxysulfate structural integrity and exposure concentrations were maintained during the 30-d treatment period, with average measured concentrations of 0.7, 1.27, 2.2, 4.31, and 10.18 mg/L. No effects were observed on the aquatic macrophyte Myriophyllum aquaticum at the highest concentration tested. A sevenfold increase in periphyton biomass at 10.18 mg/L was observed relative to controls primarily because of increases in density of the filamentous alga Mougeotia sp. Densities of the invertebrates Annelida (Stylaria), Amphipoda, Copepoda, Trichoptera (Hydropsychidae), Cladocera, and Diptera (Chironomidae) significantly decreased in streams treated with AES at 10.18 mg/L. Densities of drifting invertebrates were not observed to be affected at any concentration tested. Reproduction of Pimephales promelas significantly decreased at 1.27 mg/L and growth of juvenile Lepomis macrochirus was significantly affected at 4.31 mg/L. Multivariate cluster analysis and nonmetric multidimensional scaling ordination showed distinct structural effects on the invertebrate communities in the streams treated with AES at 10.18 mg/ L compared to the control and streams treated at < 10.18 mg/L through the 30-d treatment. Convergence of the communities treated at 10.18 mg/L toward control communities in the ordination suggests recovery in these communities after termination of surfactant treatment. The results from this study support an ecosystem value of > 2.0 mg/L, and indicate that the conservative Dutch risk assessment for AESs has at least a fivefold margin of safety.     

Influence of tetrachloroethylene on the biota of aquatic systems: Toxicity to phyto- and zooplankton species in compartments of a natural pond

This study was designed to determine the effects of tetrachloroethylene on the phyto- and Zooplankton community at initial concentrations of 1.2 and 0.44 mg/L in separated compartments of an experimental pond. Measurements in the surrounding water were made simultaneously to detect possible effects of compartmentalization. Residues as low as 0.1 mg/L could be analyzed 5 days (low dose) and 38 days (high dose) post application. In all applied biotopes, a lethal effect on the Daphnia population was detected. The phytoplankton community showed an increase of relative abundance and a decrease in species diversity. Studies of the frequency distribution of six selected phytoplankton species demonstrated the total elimination of at least four species from the treated compartments. In spite of different dosing, only weak differences could be found in toxic effects between the low and high dosed compartments. No significant chemical-induced effect was observed on the physico-chemical properties of the treated water.     

The effects of heavy metal contamination on the aquatic biota of Buttle lake and the Campbell river drainage (Canada)

Concentrations of zinc and copper in a series of large oligotrophic lakes on Vancouver Island, Canada, have been increasing since mining operations began near the south end of Buttle lake in 1966. Declines in species diversity of both phytoplankton and periphyton have occurred since the beginning of mining activity with the disappearance of metal sensitive forms such asAsterionella andRhizosolenia. Cladoceran and calanoid copepod numbers and species diversity increase as the metal concentrations decline downstream from the mine. Bioassays carried out withDaphnia pulex show increasing toxicity downstream. Metal concentrations in Zooplankton and metallothionein concentrations in rainbow trout liver are correlated to metal concentrations in the water. The water was not acutely toxic to rainbow trout in laboratory simulations of Buttle Lake conditions or duringin situ exposures (1 month). Specific sublethal deleterious effects have not yet been determined.     

Assessing structural and functional plankton responses to carbendazim toxicity

A model ecosystem experiment was conducted to investigate the ability of an in situ Daphnia magna feeding bioassay to assess impairment of ecosystem function. Animals were deployed in model ecosystems dosed with different concentrations of the fungicide carbendazim, and effects on the postexposure feeding rate of D. magna were compared with effects on zooplankton species richness (ecosystem structure) and development of phytoplankton biomass (ecosystem function). In the medium-dosed systems (21 microg/L), a structural change was observed within the zooplankton community, but no indirect effects on phytoplankton development were detected. It appears that at this treatment level, functional redundancy was sufficient to prevent functional impairment despite species loss. The feeding assay did not show any response at this concentration. In the high-dosed systems (221 microg/L), structural changes in the zooplankton community were accompanied by increased development of phytoplankton biomass. The feeding bioassay also showed a significant response at this concentration. At the high treatment level, species loss resulted in functional impairment, indicating that at this level, functional redundancy could not compensate for loss of individuals. The D. magna feeding bioassay matched well with the functional response (i.e., the indirect effects on phytoplankton) in the dosed systems but not with the more subtle effects on zooplankton community structure. These results lend positive support to the use of in situ feeding bioassays in combination with structural indices, such as species richness, to assess the effects of stress on ecosystem functioning in a direct way.     

Effect of intraperitoneally injected tocopherol on vitamin E status of dairy cow

The effect of intraperitoneal (IP) administration of vitamin E on the concentration of tocopherol in the blood and milk of cows was studied. Two trials were carried out using a total of twenty-four Holstein cows. In the first trial, two doses of dl-alpha-tocopherol (1 g and 5 g) were given to six cows in each treatment. Administration of 1 g caused a small increase in blood and milk concentrations; dosing with 5 g IP caused appreciable increases in both plasma and milk concentrations. Plasma and milk concentrations peaked 1 d after dosing with a maximum value of 10.7 micrograms/ml plasma and 1.39 micrograms/ml milk. Then there was a continuous decline during the 14 d experimental period. In the 2nd trial, twelve cows were dosed IP with the acetate form of vitamin E. Six cows were given 5 g and six others 10 g of dl-alpha-tocopherol acetate. Maximum plasma vitamin E concentrations occurred at d 2 (7.4 micrograms/ml) and d 1 (10.9 micrograms/ml) for the cows dosed with 5 and 10 g of the ester form, respectively. Milk vitamin E concentrations were significantly higher (P less than .05) during the first 4 d for cows dosed with 10 compared to those given 5 g. During the 14 d experimental period, maximum milk vitamin E concentration for the 10 g group was 141 micrograms vitamin E/g fat 2 d after dosing and for the 5 g group 62 micrograms vitamin E/mg fat also at 2 d after dosing. The technique of dosing cows with vitamin E by IP proved to be an effective way for increasing vitamin E status. These treatments had no effect on cholesterol content of milk fat. However, it was noted that cholesterol level was lower in PM milking as compared to AM milking.