Variability associated with testing cottonseed for aflatoxin

The sampling, subsampling, and analytical variance associated with testing cottonseed for aflatoxin were estimated by use of 4.54 kg samples, 100 g subsamples, and the Velasco method of analysis. Regression analysis indicated that each of the above variance components is a function of the concentration of aflatoxin in the populations tested. Functional relationships are presented to determine the sampling, subsampling, and analytical variance for any size sample, subsample, and number of analyses. Paper 4821 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607.     

Variability of aflatoxin test results

Using 12 lb samples, 280 g subsamples, the Waltking method of analysis, and densitometric procedures, the sampling, subsampling, and analytical variances associated with aflatoxin test procedures were estimated. Regression analysis indicated that each of the above variance components is a function of the concentration of aflatoxin in the population being tested. Results, for the test procedures given above, showed that sampling constitutes the greatest single source of error, followed by subsampling and analysis. Functional relationships are presented to determine the sampling, subsampling, and analytical variance for any size sample, subsample, and number of analyses.     

Aflatoxin distribution in fines and meats from decorticated cottonseed

Nine samples of fuzzy cottonseed potentially high in aflatoxin were separated into hulls, fines and meats. Assays for aflatoxin on triplicate 50-g portions from fines (small, dry particles of kernels) and meats from each of the nine samples indicated a marked concentration of toxin in the fines. On average, there was a 17-fold difference between the aflatoxin content of the fines and that of the meats; the average toxin level in fines was 4024 μg/kg and that in the meat samples was 232 μg/kg. These results indicate a potential for marked reduction in aflatoxin content of processed cottonseed meal by physical removal of fines from meats after dehulling and before processing of meats into oil and meal.     

Mutagenic potential of ammonia-related aflatoxin reaction products in cottonseed meal

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the US Department of Agriculture studied the mutagenic potential of aflatoxin reaction products following ammoniation of contaminated cottonseed meal under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled¹⁴C-aflatoxin B₁ was added to cottonseed meal that contained ca. 4000 μg naturally incurred aflatoxin B₁/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B₁ after treatment with ammonia. The radioactivity-to-weight ratio of the fractions isolated by solvent extractions and chemical and enzymic treatments was used to measure the relative concentration of an aflatoxin decontamination product. All extract fractions having a radioactivity-to-weight ratio ≥1 were tested for mutagenic activity using theSalmonella/microsome mutagenicity test (Ames test). Purified aflatoxin B₁ was mutagenic at a concentration of ca. 0.005 μg/plate. The methylene chloride extract of the ammoniated meal after Pronase digestion exhibited a similar response when 180 μg of this fraction was applied to each plate. This fraction represented 0.16% of the original added radioactivity. The other fractions produced no detectable mutagenic response at the concentrations tested (10–1000 μg/plate) onSalmonella tester strain TA100. Ammonia treatment of aflatoxin-contaminated cottonseed meal significantly decreased aflatoxin levels, and the aflatoxin decontamination products formed by the treatment had little or no mutagenic potential.     

Unavoidable low level aflatoxin contamination of peanuts

A major portion of aflatoxin contamination of peanuts probably occurs when decayed or discolored peanuts are incompletely removed by sorting. Quality control measures have been instituted in the United States to insure that unavoidable aflatoxins in consumer peanuts and peanut products do not exceed 20 μg/kg. However, low level aflatoxin contamination, from trace amounts to about 50 μg/kg in sound mature unblemished peanuts, can occur before peanuts are dug. This low level contamination is not related to high levels of Aspergillus flavus infection or to current production practices. Low level aflatoxin contamination of peanuts may be endemic, and current sorting procedures may not be effective in removing unblemished contaminated peanuts.     

Aflatoxin content of peanut hulls

The degree of aflatoxin contamination in peanut hulls was determined by analyzing inoculated hand-shelled hulls and hulls from peanuts known to contain aflatoxin. Hulls adjusted to 20% moisture, inoculated withAspergillus flavus, and incubated 7 days at 25 C supported growth ofA. flavus but not aflatoxin production. Peanuts from 20 selected Segregation III (visibleA. flavus) lots contained 13–353 ppb of aflatoxin. The machine-shelled hulls from these lots were analyzed and 3 lots contained no detectable aflatoxin, 13 lots contained 4–88 ppb and 4 lots contained >116 ppb. Aflatoxin concentrations of 53–87 ppb were detected in hulls when peanuts containing relatively high levels of aflatoxin (up to 26.8 ppm in damaged kernels) were carefully machine-shelled. Hulls from the same samples obtained by hand-shelling contained no detectable aflatoxin. When machine-shelled hulls were screened through successively smaller screens, the aflatoxin concentration of the smallest fraction (<3.18 mm) was always highest and indicated that small peanut kernels and peanut parts in the hulls actually contained the aflatoxin. Separating hulls over a 4.76 mm round-hole screen appeared to provide a means of removal of most aflatoxin in peanut hulls. No aflatoxin was found in hulls from uncontaminated peanuts.     

Effects of ammoniation on aflatoxins in rations fed lactating cows

A multifaceted cooperative research program involving industry, government and universities was initiated to determine the effects of feeding lactating dairy cows rations containing various levels of cotton-seed and cottonseed meal that had been naturally contaminated with aflatoxins. Evidence is presented that ammoniation of aflatoxin-contaminated cottonseed and cottonseed meal eliminates the aflatoxins, producing a product safe for feeding to ruminants. The aflatoxin M₁ content of milk samples of individual cows receiving rations containing (a) prime cottonseed meal, (b) aflatoxin contaminated meal, and (c) aflatoxin contaminated meal that had ammoniation treatment is reported. Data comparing results with (d) prime cottonseed, (e) aflatoxin contaminated seed, and (f) aflatoxin-contaminated seed that had ammoniation treatment are also reported. None of the milk samples from cows fed ammoniated rations contained any detectable M₁ by the modified Jacobson et al. methodology used. The sensitibity of the method in this laboratory is 0.1 μg M₁/liter of milk. Under the conditions of this study, aflatoxin M₁ levels are related to the levels of aflatoxin B₁ consumed in the diet of the cows. Conversion ratios are reported. Aflatoxin M₁ levels in the milk, relative to the time of the cows’ initial ingestion of aflatoxin B₁, the persistence of M₁ in the milk after discontinuing ingestion of B₁, and disappearance of M₁ under the conditions of the analytical methodology used relative to storage time and temperatures, are reported for liquid milk and for frozen milk. Milk containing the highest level of aflatoxin M₁ was treated with rennet. An 80:20 partion of aflatoxin M₁ was observed between curd and whey, respectively.     

Distribution of ammonia-related aflatoxin reaction products in cottonseed meal

The fate of aflatoxin during ammoniation of contaminated cottonseed meal was studied under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled¹⁴C-aflatoxin B₁ was added to 27.7 kg meal (14% moisture) that contained ca. 4000 μg naturally incurred aflatoxin B₁/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B₁ after treatment with 4% ammonia at 40 psi, 100 C for 30 min. This treatment reduced the chemically detected aflatoxin B₁ to less than 4 μg/kg. In control nonammoniated meals, 90% of the radiolabeled material was accounted for in the methylene chloride extract. Duplicate 2-kg samples of the ammoniated meal were fractionated and the distribution of radioactivity was determined. Ca. 86% of the radioactivity was detected in the meal after initial air-drying. Ca. 25% of the added radioactivity was extracted from the air-dried meal with methylene chloride and another ca. 5% was extracted from this residue with methanol. Weak acid released 3% of the added radioactivity from the residue after methanol extraction, bicarbonate released 1% and Pronase digestion, including methylene chloride extraction of the residue, accounted for nearly 19% of the total added radioactivity. Only 37% of the added radioactivity remained in the meal matrix following solvent extractions and chemical and enzymic treatments.     

prüfung von Leinfuttermitteln (Saat,Extraktionsschrot) an wachsenden Schweinen – Futterwert,Thiocyanat-und Schilddrüsenhormonstatus

Evaluation of linseed feedstuffs (ground linseed, solvent extracted linseed meal) with growing pigs – feed value, thiocyanate and thyroid hormone status. Ground linseed (Linum usitatissimum) was tested with 9 fattening pigs in a digestibility experiment. Barley (control diet) was partially replaced either by 25% or 50% ground linseed. In three periods three animals of each of the three groups (latin square) received a diet without or with 25% or 50% ground linseed. In two feeding experiments a total of 120 weaned piglets received diets either without and with 10% ground linseed or without and with 10% solvent extracted linseed meal (Exp. 2). A low apparent organic matter digestibility of 66% of ground linseed was primarily caused by 40% apparent digestibility of crude carbohydrates (crude fibre + N-free extract). In linseed groups the intake of lignin and its faecal excretion was high. It seems that the undigestible lignin is one, however not the sole reason of reduced digestibility. A relatively high metabolizable energy of 18MJ/kg linseed dry matter (DM) results from high fat content (40% of DM). Piglets receiving ground linseed tended to higher feed consumption as well as body weight gain and lower feed to gain ratio. As a tendency the consumption of diet with solvent extracted linseed meal and the daily weight gain were lower than in control (without linseed meal). Linseed feedstuffs drastically increased thiocyanate concentration of blood serum. Cyanide is released and detoxified via sulphur transferases to thiocyanate in organism.Thiocyanate impairs thyroid function, however, only in case of iodine deficiency. In case of 0.5 mg/kg diet supplementary iodine in piglet's experiments ground linseed had no effect on thyroxine and triiodothyronine concentration oi blood serum. A lower serum thyroxine concentration of piglets which were given the solvent extracted linseed meal resulted from other causes than from iodine administration (0.5 mg/kg diet).     

Sample preparation for aflatoxin assay: The nature of the problem and approaches to a solution

Cases have been reported of individual peanuts, cottonseeds or Brazil nuts so highly contaminated with aflatoxin that, for a 50 g portion to be representative of the whole, the sample preparation procedures should grind each unit to a large number of particles and distribute them uniformly throughout the sample. Assuming uniform contamination of the individual kernel, each 50 g sample should contain 1/100 of that kernel. Even though these extreme cases may be encountered only infrequently, the more usual situation still presents difficulties because of great variability in individual kernel contamination. However, if the extreme can be handled, one can expect to handle the more usual situation. Equipment and procedures to achieve this distribution goal are described. The equipment studied includes a food chopper (Hobart), a nut mill (Thomas Mills), a disc mill (Bauer), a hammer mill (Fitzpatrick Model D comminuting machine), a hammer mill designed specifically for peanut samples (Dicken’s subsampling mill), a Polytron homogenizer (Bronwill Scientific), a vertical cutter-mixer (Hobart), and a sample splitter (Jones riffle). Commodities examined were shelled peanuts and in-shell Brazil nuts, walnuts, pecans and almonds. Comminution and mixing effectiveness were determined by particle size analysis, by distribution of kernels made radioactive by neutron activation and by aflatoxin analysis of naturally contaminated products. From the results we conclude that the ultimate in sample uniformity can be achieved with a disc mill, solvent addition to obtain a fluid system and mixing and grinding of the fluid with a dispersion mixer-grinder. A practical uniformity can be achieved in a vertical cutter-mixer with less expenditure of time and effort for the commodities studied. Presented in part at the AOCS-AACC Joint Meeting, Washington, D.C., April 1968.     

Comparison of the observed distribution of aflatoxin in shelled peanuts to the negative binomial distribution

Suitability of the negative binomial distribution for use in estimating the probabilities associated with sampling lots of shelled peanuts for aflatoxin analysis has been studied. Large samples, called “minilots,” were drawn from 29 lots of shelled peanuts contaminated with aflatoxin. These minilots were subdivided into ca. 12 lb samples which were analyzed for aflatoxin. The mean and variance of these aflatoxin determinations for each minilot were determined. The shape parameterk and the mean aflatoxin concentrationm were estimated for each minilot. A regression analysis indicated the functional relationship betweenk andm to be:k=(2.0866+2.3898m) × 10⁻⁶. The observed distribution of sample concentrations from each of the 29 minilots was compared to the negative binomial distribution by means of the Kolmogorov-Smirnov test. The null hypothesis that each of the true unknown distribution functions was negative binomial was not rejected at the 5% significance level for all 29 comparisons. Journal Series Paper of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C.     

Sampling cottonseed lots for aflatoxin contamination

Large samples called “sublots” were drawn from 41 commercial lots of contaminated cottonseed. Each sublot was subdivided into twenty 5 lb samples which were analyzed for aflatoxin. The mean, median, variance, coefficient of variation, and the estimated range among the sample concentrations were computed. The results indicated that: (A) the variance among sample concentrations was large and was found to be a function of sample concentration and (B) the distribution of sample concentrations was skewed; the density of sample values was greater below the sublot concentration.     

A simplified procedure for the determination of aflatoxin B1 in cottonseed meals

A simplified procedure for the detection of aflatoxin B₁ in cottonseed meals has been developed. The procedure substantially reduces the time and cost of aflatoxin analysis. A single chromatographic column of celite is used to concentrate and purify the aflatoxin fractions. A new solvent system of ether-methanol-water (96:3:1) improves separation of aflatoxin fractions on TLC plates. This improvement permits measurement of B₁ spots in one sweep of the plate with a densitometer using a 10–15 mm slit. The number of samples which can be screened for aflatoxin contamination on one TLC plate is doubled by re-use of the plate. The use of technical reagents and stainless steel beakers also helps reduce the time and cost of analysis. The procedure is sensitive to about 5 μg of B₁ per kg of meal. Presented at the joint meeting of the AOCS-AACC April, 1968, Washington, D.C.     

Raffinose content may influence cottonseed susceptibility to aflatoxin contamination

The effect of the cotton storage trisaccharide raffinose and cottonseed storage protein (CSP) in combination on aflatoxin production by Aspergillus flavus was investigated. The ability of ground whole cottonseed and water-extracted cottonseed meal to support fungal biosynthesis of aflatoxin also was assessed in vitro. Dose response data showed that utilization of raffinose as a sole carbon source supported growth and aflatoxin production by A. flavus. When raffinose was a carbon source and CSP was the sole nitrogen source, aflatoxin levels were stimulated up to fourfold above those in raffinose reference media. Results with ground whole cottonseed as a sole carbon/nitrogen source demonstrated the capacity for aflatoxin production in A. flavus cultures. Lipid extraction of ground whole seed severely reduced aflatoxigenesis potential; however, lipid extraction followed by water extraction of ground whole seed restored much of the aflatoxin biosynthetic potential, suggesting the presence of a water soluble inhibitory factor. Accessible carbon appeared to be the limiting resource in water-extracted meal, as a raffinose supplement stimulated aflatoxin production. Either cottonseed storage lipid or raffinose was capable of providing accessible carbon to A. flavus. Raffinose and CSP in developing and mature cottonseed may predispose seed to potentially high levels of aflatoxin contamination by A. flavus upon seed infection.     

中性氧化铝柱净化-UPLC-MS/MS法测定乳和乳制品中黄曲霉毒素M_1

建立了中性氧化铝固相萃取柱净化,超高效液相色谱-串联质谱法(UPLC-MS/MS)测定乳和乳制品中黄曲霉毒素M1的分析方法。样品经乙腈提取,乙酸锌辅助沉淀蛋白,中性氧化铝SPE柱净化,以乙腈和0.1%甲酸水溶液作为流动相进行梯度洗脱,以电喷雾离子源(ESI)在正离子多反应监测(MRM)模式下进行测定,外标法定量。结果表明,黄曲霉毒素M1在0.1~50μg/L浓度范围内呈良好的线性关系,相关系数(r2)大于0.99;空白样品加标回收率在70.8%~79.2%之间,相对标准偏差小于8%。方法检出限为0.02μg/kg,定量限为0.05μg/kg。该方法实用、准确、灵敏,适用于乳和乳制品中黄曲霉毒素M1的测定。     

Use of α-tocopherol acetate to improve fresh pig meat quality of full-fat rapeseed-fed pigs

Thirty-two pigs were allocated to one of four diets, FFRD0 and FFRD200, containing full-fat rapeseed (FFR), 150 g/kg [25–50 kg liveweight (LW)], and 250 g/kg (50–90 kg LW), or CD0 and CD200, containing equivalent quantities of rapeseed meal and 34 g/kg (25–50 kg LW) or 59.2 g/kg (50–90 kg LW) coconut oil and lard (0.5:0.5, w/w). Diets FFRD200 and CD200 were supplemented with 200 mg/kg α-tocopherol acetate (ATA). ATA supplementation significantly (P<0.001) reduced muscle drip loss. The melting point (°C) of subcutaneous fat was significantly lowered by FFR (P<0.001) but increased by ATA supplementation (P<0.05). Tissue α-tocopherol (AT) concentrations were significantly increased by ATA supplementation. Longissimus dorsi AT concentration was positively correlated with AT concentration in subcutaneous fat (r=0.86) and in plasma at 35 (r=0.65) and 77 (r=0.85) days of feeding (P<0.001). In both L. dorsi and subcutaneous adipose tissue lipids, FFRD caused a significant (P<0.001) decrease in the ratio of n-6 to n-3 fatty acids and a significant (P<0.001) increase in the ratio of polyunsaturated to saturated fatty acids. AT supplementation significantly reduced the susceptibility of L. dorsi and subcutaneous fat to lipid oxidation during storage at 4°C for up to 16 d. For all dietary treatments and storage times, lipid oxidation [mg malondialdehyde (MDA)/kg muscle] was greater in the surface layer (0–2.5 mm) of L. dorsi than below the surface (2.5–5 mm). Oxidative stability of L. dorsi lipids to iron-induced lipid peroxidation was significantly improved (P<0.001) by AT supplementation. Meat from pigs fed FFRD diets was significantly less stable to iron-induced oxidation (nmoles MDA/mg protein) at the longer incubation periods (100 and 200 min). The susceptibility of L. dorsi to iron-induced lipid oxidation decreased as the ratio of the tissue concentration of AT to unsaturated fatty acid increased.     

Bright greenish-yellow fluorescence and aflatoxin in recently harvested yellow corn marketed in north carolina

Corn kernels that exhibited bright greenish-yellow fluorescence (BGYF) under long-wave ultraviolet light were hand-picked from samples of yellow corn produced in eastern North Carolina. The BGYF kernels from 113 4-kg samples contained an average of 8665 parts per billion (ppb) aflatoxin compared to an average of 46 ppb in the non-BGYF kernels. A regression analysis between the ppb aflatoxin concentration and the wt % BGYF kernels in 2,304 4.5-kg samples produced the regression equation: ppb in sample =197 (wt % BGYF). The correlation coefficient for the analysis was 0.90. Testing programs to reduce aflatoxin concentrations in purchased lots of corn based on either the BGYF method or the AOAC chemical assay method were compared. The average aflatoxin concentration in lots accepted by the AOAC method was 4 ppb, 10 ppb or 18 ppb when an acceptance level of < 20 ppb, < 50 ppb or < 100 ppb, respectively, was used. For the BGYF method, the average aflatoxin concentration in accepted lots was 10 ppb, 16 ppb or 22 ppb when an acceptance level of < 0.10% BGYF, < 0.25% BGYF or < 0.50% BGYF, respectively, was used. Approximately the same percentage of lots were accepted by both methods when either the low, medium or high acceptance level was used. Paper no. 6930 of the Journal Series of the North Carolina Agriculture Research Service (NCARS), Raleigh, NC.     

Destruction of aflatoxins in peanut protein isolates by sodium hypochlorite

Sodium hypochlorite has been tested for destruction of aflatoxins during the preparation of peanut protein isolates from raw peanuts and defatted peanut meal. The treatments were evaluated by determination of the aflatoxins in the products by thin layer chromatography. Effects of sodium hypochlorite concentration, reaction pH, temperature, and time were studied. Results show that both the sodium hypochlorite concentration and pH are important factors in reducing the concentration of aflatoxins in the protein isolates to nondetectable levels. The treatment with 0.4% sodium hypochlorite at pH 8 produced protein isolates with trace amounts of aflatoxins B₁ and B₂ from ground raw peanuts containing 725 ppb aflatoxin B₁ and 148 ppb aflatoxin B₂, whereas untreated protein isolates contained 384 ppb aflatoxin B₁ and 76 ppb aflatoxin B₂. At pH 9, 0.3% sodium hypochlorite reduced the aflatoxin B₁ content in the protein isolates from 300 ppb to below detectable quantities and the aflatoxin B₂ content from 52 ppb to 2 ppb. Similar results were obtained at pH 10 for 0.3% sodium hypochlorite concentration. In the case of defatted peanut meal which contained 136 ppb aflatoxin B₁ and 36 ppb aflatoxin B₂, 0.25% sodium hypochlorite concentration at pH 8 (0.20% at pH 9; 0.15% at pH 10) reduced both the aflatoxin B₁ and B₂ contents to below detectable quantities in protein isolates as compared to aflatoxin levels of ca. 75 ppb B₁ and 17 ppb B₂ in the untreated protein isolates. Reaction temperature and time did not affect the destruction of aflatoxins significantly.     

Effect of species and genotype on the efficiency of enrichment of poultry meat with n−3 polyunsaturated fatty acids

The effect of poultry species (broiler or turkey) and genotype (Wrolstad or BUT T8 turkeys and Ross 308 or Cobb 500 broilers) on the efficiency with which dietary long-chain n−3 PUFA were incorporated into poultry meat was determined. Broilers and turkeys of both genotypes were fed one of six diets varying in FA composition (two replicates per genotype x diet interaction). Diets contained 50 g/kg added oil, which was either blended vegetable oil (control), orpartially replaced with linseed oil (20 or 40 g/kg diet), fish oil (20 or 40 g/kg diet), or a mixture of the two (20 g linseed oil and 20 g fish oil/kg diet). Feeds and samples of skinless breast and thigh meat were analyzed for FA. Worlstad dark meat was slightly more responsive than BUT T8 (P=0.046) to increased dietary 18∶3 concentrations (slopes of 0.570 and 0.465, respectively). The Ross 308 was also slightly more responsive than the Cobb 500 (P=0.002) in this parameter (slopes of 0.557 and 0.449). There were no other significant differences between the genotypes. There was some evidence (based on the estimates of the slopes and their associated standard errors) that white turkey meat was more responsive than white chicken meat to 20∶5 (slopes of 0.504 and 0.289 for turkeys and broilers, respectively). There was no relationship between dietary 18∶3 n−3 content and meat 20∶5 and 22∶6 contents. If birds do convert 18∶3 to higher FA, these acids are not then deposited in the edible tissues.     

Evaluation of Mustard Meal as Organic Fertiliser on Tef (<Emphasis Type="Italic">Eragrostis tef</Emphasis> (Zucc) Trotter) under Field and Greenhouse Conditions

Experiments were conducted to evaluate the potential use of mustard meal as organic fertiliser on tef (Eragrostis tef (Zucc) Trotter). Mustard meal is a high quality nutrient source with 6.35% lignin, 2.1% total extractable polyphenol, C to N ratio of 14 and lignin to N ratio of 1.1. Under field conditions the effect of tef on Nitosol was studied in a split plot design with three replications. Grain yield increases due to increased mustard meal N rate varied from 2 to 116% over the control. The agronomic efficiency was 3.0, 8.3 and 13.5 kg when N was applied at 15, 23 and 31 kg ha⁻¹, respectively. The mustard meal N use efficiency was 7.6, 20.6 and 33.7% for the above-indicated N rates. Application of mustard meal in powder form was more effective than granular. In the greenhouse, the effect of mustard meal and urea N mixed in different quantity was studied with ¹⁵N technique. The N derived from fertiliser was lowest (3.5%) when 20 mg pot⁻¹ from urea was combined with 100 mg pot⁻¹ from mustard meal and highest (11%) when 67 and 33 mg pot⁻¹ as urea and mustard meal were combined, respectively. The N derived from mustard meal was lowest (3.3%) when mustard meal and urea N were combined at 50 mg N pot ⁻¹ each, and highest (8.9%) when combined at 20 and 100 mg N pot⁻¹, respectively. The urea and mustard meal N yields significantly varied between the treatments. The N use efficiency from urea (FNUE) varied from 38.4 to 43%. Combining urea and mustard meal N at 50 mg N pot⁻¹ each has decreased FNUE to 4.4% compared to the urea N applied alone at 50 mg N pot⁻¹. N use efficiency (NUE) from mustard meal was highest (38.4%) when mustard meal and urea N were combined at 33 and 67 mg N pot⁻¹, respectively, and lowest when it was combined at 67 and 33 mg N pot⁻¹.