Liang & Yang 2019 — Cu/Zn combined-stress effects on antioxidant enzymes (CAT, POD, SOD), MDA, and soluble protein in olive leaves (Sichuan, China)

Liang and Yang conducted a 40-day pot experiment on annual Olea europaea L. cv. ‘Foao’ cutting seedlings in Xichang, Sichuan, exposing them to four combined Cu/Zn soil-spike treatments and a zero-dose control to characterise the antioxidant-system response to the two heavy metals jointly. Foliar catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), malondialdehyde (MDA), and soluble protein were measured at the end of the treatment period. The study is a phytotoxicity/stress-tolerance experiment using applied-soil concentrations far above ambient agricultural exposures; it does not measure Cu, Zn, or any other metal concentration in olive fruit, olive oil, or any consumable matrix. Its contribution to the Heavy Metal Index corpus is mechanistic context for how the olive plant responds to high Cu/Zn co-exposure at the leaf-tissue level, not a contamination data point for olive-derived foods.

Key numbers

Soil-spike design (Table 1, p. 2). Five treatments, each in triplicate measurement on n=10 pots:

• CK (control): Zn²⁺ 0 mg/L; Cu²⁺ 0 mg/L
• A: Zn²⁺ 150 mg/L; Cu²⁺ 50 mg/L
• B: Zn²⁺ 250 mg/L; Cu²⁺ 150 mg/L
• C: Zn²⁺ 300 mg/L; Cu²⁺ 250 mg/L
• D: Zn²⁺ 325 mg/L; Cu²⁺ 350 mg/L

These are soil-applied concentrations of the spike solutions, not measured tissue concentrations. Treatment duration: 40 days.

Catalase (CAT) activity in olive leaves (Table 2, p. 3; units not stated in source — likely U/g FW or U/mg protein, mean±SD, p<0.05 significance letters in last column):

• CK: 116.667 ± 4.226 (d)
• A: 76.167 ± 4.475 (e) — 34% decrease vs CK at the lowest Cu/Zn dose
• B: 154.667 ± 9.208 (c) — 32.6% increase vs CK
• C: 195.000 ± 8.322 (b) — 67.1% increase vs CK
• D: 236.500 ± 11.056 (a) — 103.06% increase vs CK at the highest Cu/Zn dose

Peroxidase (POD) activity in olive leaves (Table 3, p. 4):

• CK: 47.330 ± 1.333 (c)
• A: 26.000 ± 3.055 (d) — 45.38% decrease vs CK
• B: 53.330 ± 3.528 (c) — 12.7% increase vs CK
• C: 92.670 ± 3.712 (b) — 95.8% increase vs CK
• D: 171.330 ± 5.812 (a) — 262% increase vs CK (3.62× control; paper text reports the maximum-vs-minimum increase as 56.57% which appears to refer to a different comparison, see Verification notes)

Superoxide dismutase (SOD) activity in olive leaves (Table 4, p. 4):

• CK: 117.176 ± 1.334 (c)
• A: 173.277 ± 1.763 (a) — 47.9% increase vs CK (SOD peak)
• B: 146.803 ± 5.414 (b) — 25.3% increase vs CK
• C: 91.076 ± 4.070 (d) — 22.3% decrease vs CK
• D: 46.390 ± 2.098 (e) — 60.4% decrease vs CK at the highest Cu/Zn dose

Malondialdehyde (MDA), a lipid peroxidation marker, in olive leaves (Table 5, p. 5; units not stated in source):

• CK: 0.092 ± 0.005 (d)
• A: 0.115 ± 0.009 (d) — 25% increase vs CK
• B: 0.151 ± 0.010 (c) — 64.13% increase vs CK
• C: 0.255 ± 0.012 (b) — 177.17% increase vs CK
• D: 0.367 ± 0.012 (a) — 298.91% increase vs CK (3.99× control)

Soluble protein in olive leaves (Table 6, p. 5):

• CK: 0.069 ± 0.000 (d)
• A: 0.076 ± 0.001 (c) — 10.14% increase
• B: 0.083 ± 0.004 (b) — 20.29% increase
• C: 0.089 ± 0.000 (b) — 28.99% increase
• D: 0.104 ± 0.001 (a) — 50.72% increase vs CK

Trend summary across the four-dose Cu/Zn gradient:

• CAT: U-shape — drops at the lowest spike, then climbs monotonically to ~2× control at the highest spike.
• POD: U-shape — drops at the lowest spike, then climbs monotonically to ~3.6× control at the highest spike.
• SOD: inverted U-shape — rises at low spike, peaks at A, then declines steeply to <0.4× control at the highest spike.
• MDA: monotonic increase from CK to D (~4× control).
• Soluble protein: monotonic increase from CK to D (+50.7%).

Methods

Plant material: annual Olea europaea L. cv. ‘Foao’ cutting seedlings (height ~55-65 cm), leaves originally collected in 2016 from Lizhou Town, Xichang City. 50 pots (3 kg soil each), assigned to 5 treatments × 10 replicates.

Experimental site and timing: Lizhou Town, Xichang City, Sichuan, September-December 2016.

Heavy metal stress treatment: ZnSO₄·7H₂O and CuSO₄·7H₂O solutions applied to the soil at the four dose combinations in Table 1. (The methods text also mentions FeSO₄·7H₂O in the reagent list and the introduction repeatedly refers to “Cu and Zn” stress, with isolated text passages erroneously naming “Fe” instead of “Cu” — see Verification notes inconsistency 1.) Treatment duration: 40 days.

Tissue preparation: ~0.3 g leaf tissue per sample, ground in pre-cooled mortar with 2 mL pH 7.8 phosphate buffer and a small amount of quartz sand into a homogenate; rinsed up to 6 mL final volume with pH 7.8 PBS; centrifuged at 15,000 g for 10 min at 4 °C; supernatant stored on ice in the dark until assay.

Enzyme and biochemistry assays:

• CAT: hydrogen peroxide (H₂O₂) decomposition method.
• POD: guaiacol method.
• SOD: nitroblue tetrazolium (NBT) photoreduction method (with L-methionine and riboflavin as system components per the reagent list).
• MDA: thiobarbituric acid (TBA) method (paper text says “arsenic barbituric acid (TBA)” — this appears to be a translation artefact for thiobarbituric acid).
• Soluble protein: Coomassie Brilliant Blue staining (Bradford-style).

Soluble sugar is mentioned in the methods reagent list and Determination Method paragraph but not reported in the Results section.

Instrumentation: enzyme meter (microplate/UV reader), HH-8 digital thermostat water bath, centrifuge, spectrophotometer. Vendor models not specified.

Statistics: mean ± SD across 3 measurement repetitions; Microsoft Excel for descriptive statistics; IBM SPSS Statistics v19 for ANOVA / significance testing; significance lettering at p<0.05 (same-letter treatments not significantly different).

Analytical scope: the paper measures leaf-tissue antioxidant-system biomarkers (CAT, POD, SOD, MDA, soluble protein) under combined Cu²⁺/Zn²⁺ soil exposure. It does not measure Cu or Zn concentrations in olive leaves, olive fruit, or olive oil, and it does not measure any other heavy metal. No speciation is performed (the spiking compounds are CuSO₄ and ZnSO₄, so the bioavailable form is Cu²⁺ and Zn²⁺). The study has no consumer-exposure component.

Implications

Certification: Not relevant to certification-threshold setting. The paper does not produce contamination data in olive or olive oil that could be pooled into a percentile distribution for HMTc rows. It is mechanistic phytotoxicity context for the olive crop, not a measurement of food-system contamination.

Courses: Usable as a pedagogical example of the inverted-U (hormesis-like) response of SOD activity to heavy-metal stress in plants and of the monotonic increase of MDA (membrane lipid peroxidation) and soluble protein (osmotic-adjustment response) under combined Cu/Zn exposure. The CAT/POD U-shape is also a recurring pattern in plant heavy-metal stress physiology.

App: Not relevant to consumer-app contamination-likelihood scoring for foods. The applied soil concentrations (50-350 mg/L Cu²⁺ and 150-325 mg/L Zn²⁺ in solution form) are orders of magnitude above ambient agricultural soils.

Speciation: Cu²⁺ and Zn²⁺ as sulfate-salt soil spikes. No fractionation or speciation analysis.

Wiki pages this source may touch

• olives (phytotox/stress-physiology context; no fruit-tissue contamination measured)
• olive-oil (phytotox/stress-physiology context; no oil contamination measured)
• copper
• zinc

Verification notes

• 2026-06-01 ingest (Claude Opus 4.7, MFK_article-2): fresh page from the Manual Fetch Kimi / June 1 batch. Three identity checks (DOI 10.1088/1755-1315/300/5/052058 — no hit; raw_handle MFK_article-2 — no hit; cite-key liang-yang2019-olive-leaves-cu-zn-stress — no hit; surname grep liang2019 returned only liang2019-beijing-foodstuffs-health-risk.md which is a different first author Liang G., different DOI 10.3390/ijerph16060909, different topic). No merge-enhance target; NEW path.
• Cite-key disambiguation: the existing liang2019-* is Liang G., Gong W., Li B., Zuo J., Pan L., Liu X. (Beijing foodstuffs health risk, IJERPH). This paper is Liang Jian + Yang Weifei (Xichang olive-leaf phytotox). Cite-key includes second-author surname (liang-yang2019-...) to make the distinction visible in source-list scans without relying solely on the topic suffix.
• License: the PDF page 2 explicitly states “Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence” with a CC BY logo. Recorded as CC BY 3.0.
• DOI assigned by IOP Publishing: 10.1088/1755-1315/300/5/052058 (visible in the running header on every page).
• Conference: REES2019 (2nd International Conference on Resources and Environmental Engineering Sustainability), proceedings published in IOP Conf. Ser.: Earth Environ. Sci. Vol. 300. Conference-proceedings status is the basis for evidence_tier C.
• Source type: peer-reviewed (IOP Conference Series is editorially peer-reviewed; not a primary research journal but not a preprint).
• Evidence tier C rationale: conference-proceedings publication (not a primary journal); small experimental scope (one cultivar, one location, one season, 40-day duration); single dose-response curve; multiple translation-quality issues throughout the paper text (machine-translation artefacts visible in many sentences); phytotoxicity-mechanism study with no direct food-system contamination measurement; n=10 pots per treatment is adequate for ANOVA but modest. Reasonable as mechanistic context, not as primary contamination evidence.
• metals: [Cu, Zn] reflects the analytes that were applied as soil stressors and that the antioxidant-system response is attributed to. The paper does not report Cu or Zn tissue concentrations. The wiki’s Cu/Zn slugs are the appropriate abbreviation tags; speciation is implicit (Cu²⁺ and Zn²⁺ from sulfate salts).
• products: [] (empty) is intentional. The paper measures plant leaves, not any consumable product (olive fruit, olive oil, etc.). Per CLAUDE.md Part 5b, frontmatter should reflect the actual scope of the source; over-specifying products: ["[[products/olive-oil]]"] would route this phytotox study onto the olive-oil product page as direct evidence, which it is not. Routing-discipline audit --warn-only may flag the empty products field; this is a phytotox study not a food/personal-care contamination paper, so the warning (if it fires) is a false positive in the audit’s intended scope.
• ingredients: [olives, olive-oil] is included because the routing layer’s role is to surface this paper as phytotox/stress-physiology context on the olive and olive-oil ingredient pages. The synthesis pass for those pages may treat this source as exposure_only or broad_product_context rather than direct evidence.
• matrices uses bare-string descriptors (not wikilinks): olive-leaves, soil-spike-experiment, plant-tissue. These convey “this is a leaf-tissue assay under a soil-spiking experimental design” without overspecifying a product/ingredient routing destination.
• jurisdictions: [CN] — single-country study, Sichuan Province.
• Sample size: 50 pots total (5 treatments × 10 replicates). Foliar physiological-index measurements repeated 3 times per treatment. sample_n: 50 reflects experimental units; the 3× measurement repetition is captured in the methods narrative.
• Paper-internal inconsistency 1 (Fe vs Cu naming): the introduction and one parenthetical in the abstract refer to “heavy metal iron and Zn” and “Fe²⁺ and Cu²⁺ resistance,” but Table 1, the methods reagent list, and every Results/Discussion table all consistently refer to Cu²⁺ + Zn²⁺. The Fe references appear to be machine-translation artefacts (the Chinese characters for Fe/Cu may have been mis-translated in places). The Methods reagent list also includes FeSO₄·7H₂O, but no Fe data is reported and Table 1 shows only Zn and Cu spikes. The wiki page treats the experiment as Cu/Zn combined stress (matching Tables 1-6) and flags the Fe references as translation artefacts.
• Paper-internal inconsistency 2 (POD increase percentage): the Results §3.2 text states “the activity reached the maximum at Cu350+Zn325, which increased by 56.57%” but the table values give D = 171.330 vs CK = 47.330, an increase of 261.99% (3.62× control). The same paragraph also writes that POD content under A, C and D treatments was “0.55, 1.96 and 3.62 times of the control, respectively” — these multipliers match the table (26/47.33 = 0.549; 92.67/47.33 = 1.958; 171.33/47.33 = 3.621). The “56.57%” figure does not reconcile with any other quantity reported for POD and appears to be a transcription error or a reference to a different sub-quantity. The wiki page reports the table-derived percentages.
• Paper-internal inconsistency 3 (CAT trend description in Conclusion §4.1.1): the discussion text says “the activity values of CAT and POD in the leaves showed a tendency to decrease first and then increase in the experimental concentration range.” This matches the tables. The abstract says “CAT and POD … showed a trend of decreasing first and then increasing” — consistent. No inconsistency to flag; this confirms the U-shape pattern.
• Paper-internal inconsistency 4 (MDA last-treatment description): Discussion mentions “MDA content of olive oil increased by 43.92% under D treatment” — this 43.92% figure does not match the table-derived 298.91% increase from CK = 0.092 to D = 0.367. The “43.92%” appears to be a leftover figure from an earlier draft or a different comparison (perhaps D vs C: 0.367/0.255 = 1.439, i.e., 43.92% above C — which matches). The page reports the CK-based percentages from the table.
• Paper-internal inconsistency 5 (SOD activity decrease vs CK): Results §3.3 says “The enzyme activities of treatment C and treatment D were 77.73% and 39.60%, respectively.” 91.076/117.176 = 0.7773 (77.73% of CK) and 46.390/117.176 = 0.3960 (39.60% of CK). These match the table — the paper is reporting percent-of-control, not percent-change. No inconsistency; clarified in the Key numbers section by reporting decreases relative to CK (22.3% decrease for C, 60.4% decrease for D).
• Units omitted by source: the paper does not state activity units for CAT, POD, or SOD in the tables or methods (likely U/g FW or U/mg protein based on the standard assay protocols, but the source does not specify). MDA and soluble protein values are also given without explicit units (likely µmol/g FW for MDA and mg/g FW for soluble protein based on conventional plant-physiology assay outputs, but again the paper does not state). The wiki page reproduces the values as given without inferring units.
• TBA assay name typo: the methods text spells “arsenic barbituric acid (TBA)” — this is a translation artefact for thiobarbituric acid (TBA). The TBA reagent in the reagent list is correctly named.
• Brand firewall (CLAUDE.md Part 12): the paper does not name any brand, mill, or commercial product. The plant material is described by cultivar (‘Foao’) and origin (Lizhou Town nursery). No brand-firewall edits required.
• Wiki/HMTc firewall (CLAUDE.md Part 2): no synthesis claims, no HMTc threshold references, no consumer risk advisories were drafted into this page. The Implications section frames the paper as mechanistic phytotox context with explicit “not relevant to” caveats for certification, app, and consumer-exposure use cases.
• Slug check against taxonomy snapshot (2026-05-18): olives and olive-oil exist in ingredients; copper and zinc exist in metals. The page uses Cu and Zn in the frontmatter metals: array per the abbreviation vocabulary, and [[metals/copper]] / [[metals/zinc]] in the bottom-of-page wikilinks section per the page-slug convention.

Page history

The five most recent substantive edits to this page. The full version history lives in git; when DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.