. Author manuscript; available in PMC: Jun 10. Published in final edited form as: Ann Emerg Med. Sep;46(3):275'284. doi: 10./j.annemergmed..03.016

Intentional self-poisoning with the chlorophenoxy herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA)

Darren M Roberts

Darren M Roberts, MBBS

(1) South Asian Clinical Toxicology Research Collaboration (SACTRC), Medical School, Australian National University, Australia (2) SACTRC Clinical Units, North Central Province, Sri Lanka Find articles by Darren M Roberts (1),(2), Ruwan Seneviratne

Ruwan Seneviratne, MBBS

(2) SACTRC Clinical Units, North Central Province, Sri Lanka Find articles by Ruwan Seneviratne (2), Fahim Mohammed

Fahim Mohammed, B Pharm

(2) SACTRC Clinical Units, North Central Province, Sri Lanka Find articles by Fahim Mohammed (2), Renu Patel

Renu Patel, AssDipAppSc

(3) Queensland Health Scientific Services, Coopers Plains, Queensland, Australia Find articles by Renu Patel (3), Lalith Senarathna

Lalith Senarathna, BSc

(2) SACTRC Clinical Units, North Central Province, Sri Lanka Find articles by Lalith Senarathna (2), Ariyasena Hittarage

Ariyasena Hittarage, MD MRCP

(4) Anuradhapura General Hospital, Anuradhapura, Sri Lanka Find articles by Ariyasena Hittarage (4), Nick A Buckley

Nick A Buckley, MD FRACP

(1) South Asian Clinical Toxicology Research Collaboration (SACTRC), Medical School, Australian National University, Australia Find articles by Nick A Buckley (1), Andrew H Dawson

Andrew H Dawson, MBBS FRACP

(2) SACTRC Clinical Units, North Central Province, Sri Lanka (5) Faculty of Medicine, University of Peradeniya, Sri Lanka Find articles by Andrew H Dawson (2),(5), Michael Eddleston

Michael Eddleston

(2) SACTRC Clinical Units, North Central Province, Sri Lanka (6) Department of Clinical Medicine, Faculty of Medicine, University of Colombo, Sri Lanka (7) Department of Tropical Medicine, Oxford University, United Kingdom Find articles by Michael Eddleston (2),(6),(7)

(1) South Asian Clinical Toxicology Research Collaboration (SACTRC), Medical School, Australian National University, Australia (2) SACTRC Clinical Units, North Central Province, Sri Lanka (3) Queensland Health Scientific Services, Coopers Plains, Queensland, Australia (4) Anuradhapura General Hospital, Anuradhapura, Sri Lanka (5) Faculty of Medicine, University of Peradeniya, Sri Lanka (6) Department of Clinical Medicine, Faculty of Medicine, University of Colombo, Sri Lanka (7) Department of Tropical Medicine, Oxford University, United Kingdom PMCID: PMC  EMSID: UKMS  PMID: The publisher's version of this article is available at Ann Emerg Med

Abstract

Study Objective: Data on poisoning with MCPA (4-chloro-2-methylphenoxyacetic acid) is limited to six case reports. Our objective is to describe outcomes from intentional self-poisoning with MCPA in a prospective case series of 181 patients presenting to hospitals in Sri Lanka.

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Methods: Patient information was collected by on-site study doctors as part of an ongoing prospective cohort study of poisoned patients. History, clinical details and blood samples were obtained prospectively.

Results: Overall clinical toxicity was minimal in 85% of patients, including mild gastrointestinal symptoms in 44% of patients. More severe clinical signs of chlorophenoxy poisoning reported previously such as rhabdomyolysis, renal dysfunction and coma also occurred, but were uncommon. Eight patients died (4.4%). Most deaths occurred suddenly from cardiorespiratory arrest within 48 hours of poisoning; the pathophysiological mechanism of death was not apparent. The correlation between admission plasma MCPA concentration and clinical markers of severity of toxicity (physical signs, symptoms and elevated creatine kinase) was poor.

Conclusions: Intentional self-poisoning with MCPA generally causes mild toxicity, but cardiorespiratory arrest and death may occur. All patients should receive routine resuscitation and supportive care. It seems reasonable to correct acidosis and maintain an adequate urine output, but there is insufficient evidence to support other specific interventions. Our data do not support a clinical role for measurement of plasma MCPA in the acute management of poisoning and insufficient data were available to fully examine the utility of measured electrolytes and creatine kinase.

Introduction

Background

It is estimated that over 300,000 people die from pesticide poisoning each year in Asia and the Western Pacific.(1) Sri Lanka has a major problem with intentional self-poisoning, with high total and youth suicide rates.(2) The mortality from intentional self poisoning in Sri Lanka is high given ready access to potent pesticides by the largely rural population. Most poisoning deaths in Sri Lanka are due to organophosphorus (OP) pesticides, but deaths from intentional self-poisoning with other pesticides, such as chlorophenoxy herbicides are noted.(3) Toxicity from chlorophenoxy herbicides is also important in developed countries such as the United States where it is one of the most commonly used classes of pesticides.(4)

The most common chlorophenoxy herbicides are presented in Figure 1. The human literature on acute chlorophenoxy poisoning is limited to small case series and single reports of severe toxicity.(5) A third of these cases died and many others had clinical features of severe toxicity (Table 1). Most of these were reports of poisoning with 2,4-D. The selection of more serious cases for publication may mean that this does not represent the usual toxicity from chlorophenoxy poisoning nor an accurate estimate of their lethality. This is important given that some chlorophenoxy herbicides appear to have different mechanisms of toxicity and clinical features.(6-8)

Table 1.

Gastrointestinal Commonly oral burning, vomiting, abdominal pain, diarrhoea; rarely haemorrhage. Neuromuscular Skeletal muscle effects include muscle weakness, fasciculations, hyper- or hyporeflexia, ataxia, nystagmus, and myotonia which may progress to rhabdomyolysis. Both myopathic and neuropathic changes have been noted on neurophysiology testing. Occasionally prolonged agitation, confusion, coma, miosis, convulsions and nystagmus. Cardiovascular Tachycardia and hypotension Respiratory Hyperventilation with respiratory alkalosis may occur early in the poisoning, or later secondary to metabolic acidosis. CNS depression and respiratory muscle weakness may cause hypoventilation. Other effects Metabolic acidosis, pyrexia, renal failure, and electrolyte changes (particularly hypocalcaemia and hypokalaemia). Death Autopsy has revealed congestion and oedema of lungs, liver, kidneys, adrenals and brain. Early necrosis of the liver and kidneys has also been reported. The mechanism of death has not been confirmed.

Importance

MCPA (4-chloro-2-methylphenoxyacetic acid) is a widely used chlorophenoxy herbicide. It is the most important cause of chlorophenoxy poisoning in the North Central Province (NCP) of Sri Lanka.(3) There is limited information on clinical toxicity from MCPA poisoning, with only six single case reports in the literature.(9-14)

Goals of this investigation

We describe the clinical outcomes of MCPA poisoning from data gathered during a large, prospective case series of patients who presented to three Sri Lankan hospitals with a history of intentional self-poisoning.

Methods

Study design

This was a prospective descriptive study of the exposure history and clinical details on consecutive patients admitted to three hospitals in Sri Lanka (Figure 2).

Setting

Patients presenting to three of the General Hospitals in Sri Lanka between 2nd April and 27th December with a history of MCPA poisoning were included. Resources are limited in these hospitals, particularly monitored beds in the ICU and wards and laboratory facilities. Blood tests are not usually obtained in most poisoned patients. All patients were stabilized in the ward and managed with supportive care. Patients with severe poisoning were admitted to ICU if beds were available.

Selection of participants and intervention:

All patients presenting to study hospitals with self poisoning were included. A large subset of these patients were part of a randomised controlled trial (RCT) evaluating the efficacy of activated charcoal (ISRCTN). All patients with a history of acute poisoning were eligible for inclusion in this RCT, except for those who are under the age of 14 years, known to be pregnant or reported ingestion of either hydrocarbons alone or corrosives. Written informed consent was obtained prior to enrolment. Ethics approval was obtained from the University of Colombo, Sri Lanka and Oxford, United Kingdom.

Convenience blood samples were obtained on admission from 91 of the 113 patients recruited to the RCT between 8th May and 21st September for quantification of the plasma MCPA concentration using gas chromatography mass spectroscopy. These samples were in addition to those obtained by the treating physician for clinical indications.

In order to examine for the presence and time course of myotoxicity, serial convenience samples of blood were also obtained in 12 patients for measurement of creatine kinase (CK) activity. These patients presented to one hospital early in the series and were selected only because of the history of MCPA poisoning. CK activity was measured only in the 10 patients who tested positive to MCPA on admission. The medical chart of these patients was manually reviewed to correlate clinical features with plasma concentrations of MCPA and CK activity. Biochemical analyses were conducted by Queensland Health Scientific Services, Australia.

Treatment was determined by the treating physician and their team independent of the research team and irrespective of enrolment in the activated charcoal RCT. Supportive care with IV fluids, respiratory support, and inotropes were given where necessary, however alkalinisation was not used.

Data collection and analysis:

Important demographic details, including the type of poison and time since ingestion were collected on admission. Pre-determined demographic data and details of major clinical outcomes were prospectively recorded by on-site full time trained study doctors for every patient until discharge or death. These were entered directly into a hand-held computer data base at the time of recruitment and with each clinical review and evaluated by a frequency analysis. Other qualitative data were obtained directly from the medical chart for patients who died and those who provided serial blood samples.

Results

A history of MCPA poisoning was reported in 181 patients, (145 of these patients were also recruited to the RCT of charcoal). Time to hospital admission and outcomes of poisoning are listed in Table 2. Ten patients received endotracheal intubation and ventilation for respiratory failure. The incidence of spontaneous vomiting was reviewed at one the study hospitals (accounting for the 63% of all admissions), and was noted to occur in 44% of patients.

Table 2.

N (total = 181) (%) Time to presentation Unknown 1(0.5) <2 hours 18 (9.9) 2 to < hours 61 (33.7) 4 to <7 hours 62 (34.2) 7 to <12 hours 24 (13.2) 12 to <24 hours 9 (5.0) >24 hours 6 (3.3) Glasgow Coma Score (GCS) On arrival Lowest during admission 15 154 (85.1) 153 (84.5) 13-14 11 (6.1) 8 (4.4) 10-12 9 (5.0) 9 (5.0) 4-9 4 (2.2) 6 (3.3) 3 3 (1.6) 5 (2.8) Lowest recorded systolic blood pressure during admission (mmHg) >120 46 (25.4) 101-120 103 (56.9) 80-100 21 (11.6) <80 Nil Not recorded (patient alert & interactive) 11 (6.1) Outcome at Discharge Alive 173 (95.6) Dead 8 (4.4) Median time to live discharge, days (range 2.0 (1-7)

Eight patients died (4.4%, Table 3). Seven of the eight patients died within 24 to 48 hours of poisoning from cardiorespiratory arrest of an unclear pathophysiological mechanism. A delay in the time to hospital admission did not appear to contribute to this outcome. One patient (Case 5) had bilateral crepitations and purulent secretions consistent with aspiration pneumonia. The medical chart could not be located for one patient (Case 8). Oliguria or dark-coloured urine was noted in five patients. Test bolus doses of atropine were given to some patients on admission who were initially suspected of being poisoned with anticholinesterase pesticides. This was generally not continued, but may have contributed to symptoms in some patients on admission.

Table 3.

Patient Age/sex Time to hospital presentation (hours) Clinical features at presentation Treatment Features prior to death Time to death (hours) 1 48M 4 Coingestion of alcohol.RR 22-26/min, HR 104, BP 120/80 mm Hg, GCS 7/15, vomiting, miosis,epistaxis, oliguria despite fluids MDAC, intubation,IVF, GL, atropine bolus antibiotics,ICU HR=160/min, BP normal, then cardiorespiratory arrest 2 hours post admission, failed resuscitation 6 2 48M 3 Coingestion of alcohol RR 18/min, HR 80/min, BP 130/80 mm Hg, GCS 3/15, vomiting, miosis, facial flushing. SDAC, Intubation,IVF, atropine BP 70/50 mm Hg, HR 96/min, then bradycardia and CRA, failed resuscitation 6 3 28F 1 HR 90/min, BP 110/70 mm Hg, GCS 15/15, vomiting, oral secretions IVF, GL BP 110/70 mm Hg, HR 96/min,GCS 15, temp 40.4oC, B/L crepitation, cardiorespiratory arrest one hour later, failed resuscitation. 24 4 37M 5 Coingestion of alcohol HR 80/min, BP 130/80 mm Hg,GCS 10/15, severe haematemesis, aspiration with bilateral crepitations, excess oral secretions, epistaxis (?trauma) 10 hours: febrile, HR 120/min, miosis, BP 100/70 mm Hg, GCS 6/15, dark urine SDAC, ETT, IVF,antibiotics,furosemide 20 hours: RR 30/min, HR 140/min, GCS 6/15, oliguria, Urea 65, serum electrolytes normal 24 hours: RR 40/min, SpO2 92%, HR 140/min GCS 5/15. 27 hours:cardiorespiratory arrest, resuscitated, 'laboured breathing',SpO2'80% despite high flow oxygen, HR 180/min, BP 90/60 mmHg, febrile, oral bleeding. Urea 30 115, SE normal. NaHCO3 bolus,furosemide and inotropes commenced 29 hours: multiple cardiorespiratory arrests with non-sustained resuscitation. 5 28F ? Day 1 HR 80/min, BP 100/70 mmHg, GCS 11/15, nausea, vomiting, diarrhoea,abdominal pain MDAC, GL, IVF,furosemide,antibiotics,inotropes,NaHCO3, atropine Day 2: Laboured breathing,widespread crepitations, BP 95/70 mm Hg, HR 64/min, oliguria.Later in day: Respiratory arrest, 'yellow secretions' from ETT, HR <48 GCS 11/15, vomiting, diarrhoea. B/L crepitations and rhonchi, oral secretions,cyanosis bolus 30/min, thencardiorespiratory arrest, failed resuscitation. 6 50M 4 HR 110/min, BP 120/80 mm Hg, GCS 15/15, oral secretions, fasciculations and diaphoresis. Later that day became confused and agitated MDAC, GL, IVF,furosemide,atropine bolus. 20 hours: cardiorespiratory arrest, 21 hours: asystolic arrest, failed resuscitation. 20 7 45M 10 Co-ingestion of alcohol HR 80/min BP 100/40 mm Hg, GCS 3/15, oral secretions, BUN 92 ETT, IVF, atropine bolus, GL Day 2: NaHCO3 Day 2: RR 28/min, tidal volume 500 mL, HR 120/min, GCS 8/15,Temp 101.4°F, 'dark urine' hrs: RR 40/min, HR 120/min, BP 70/30 mm Hg, GCS 6/15 hrs: CRA, failed resuscitation <48 8* 49F 4 Asymptomatic, GCS 15/15, systolic BP 130mmHg SDAC Cardiorespiratory arrest 30

Of the admission bloods available for analysis, MCPA was not detected in 11/91 patients (detection limit 0.05mg/kg plasma), consistent with minimal or no exposure to MCPA. One of these patients presented with a Glasgow Coma Score (GCS) of 15/15 six hours post-ingestion, but soon developed seizures and abdominal pain. The cause of these symptoms is unknown, but most likely represents poisoning with a substance other than MCPA, for example fipronil. The patient was discharged without complication four days later. All other patients with an undetectable MCPA level developed no signs of poisoning.

The relationship between admission plasma MCPA concentration, time since ingestion and severity of toxicity appears poor (Figure 3), although only one death (Case 6) was captured in this cohort, given the difficulty in obtaining consent from patients with an altered level of consciousness on presentation. Although CK activity tended to be increased in patients with higher admission MCPA concentrations (>300mg/kg), the correlation appears poor (Table 4 and Figure 4). With the exception of Cases B and C, CK activity was only marginally elevated in these patients. Case B presented with GCS 10/15 and profuse vomiting. Myalgias and weakness developed the following day and resolved within 48 hours. Case C presented with mild haematemesis, diarrhoea, abdominal pain and anuria despite fluid loading. Urine output returned by the next day and despite some 'throat pain', the gastrointestinal symptoms had settled. He complained of generalised myalgias, but appeared otherwise well and was discharged within 50 hours. No other investigations were ordered during this admission. The CK increased until discharge (Figure 4), which together with the myalgias, may be consistent with ongoing skeletal muscle toxicity.

Table 4.

Case (age,sex) Admission plasma MCPA (mg/kg) Peak CK activity (U/L)* Severity of toxicity§ Admission MCPA concentration >300mg/kg A(27,M) 672 402 Low B (26,F) 569 942 Severe C (19,M) 591 Moderate D (39,M) 387 338] Mild E (25,F) 415 493 Mild F (27,M) 340 NE Low Admission MCPA concentration <20mg/kg G (21,M) 16 393 Low H (25,M) 17 272 Low I (38,F) 0.8 NE Low J (16,M) <0.05 NE Low

Limitations

This study was performed in secondary referral hospitals in rural areas of Sri Lanka. They have very limited resources and laboratory tests are not routinely performed. Monitored beds in the ICU or wards are not routinely available. The patient-doctor and patient-nurse ratios are high, so data on clinical features exceeding those obtained for the purpose of our study are limited. Most deaths occurred in unmonitored beds in the absence of blood tests, which limits our ability to determine the mechanism of death.. Further, as blood tests were only taken on those who consented to enrolment in the RCT, patients who were most unwell were less likely to have MCPA blood concentrations measured.

Discussion

This is the first published prospective case series of major outcomes from chlorophenoxy self-poisoning. Mild gastrointestinal symptoms were noted, but overall, toxicity from MCPA poisoning was low and mortality was less than other common pesticide poisonings presenting to the same hospitals. Although animal data suggest that other chlorophenoxy herbicides are more toxic than MCPA (Figure 1), it seems unlikely that a third of poisonings with other chlorophenoxy herbicides will be fatal. The case series and single reports that this outcome was based on were probably published because of their severity or unusual presentation, representing a reporting bias.(5)

Clinical toxicity

Variable clinical features have previously been reported for MCPA poisoning, but all are consistent with those described in our patients (Table 1). We found that 85% of patients had minimal toxicity (despite a large reported exposure in some) and mortality was 4.4%. This in-hospital death rate is higher than that noted from medications, but lower than that of other pesticides including the herbicides glyphosate (5%) and paraquat (60%) and organophosphorus insecticides (11%).(15) Blood levels confirmed ingestion of MCPA in 88% of samples tested and there was only one patient where the combination of laboratory investigations and clinical features suggested that MCPA was not responsible for the clinical presentation despite a history of ingestion. Therefore these data should be an accurate reflection of the clinical course from acute intentional self poisoning with MCPA.

Other clinical features of MCPA poisoning were not systematically recorded in each patient, but abdominal pain, myalgia, fasciculations, throat pain and miosis were noted by study doctors in some patients.

Seven of eight deaths were due to a cardiorespiratory arrest of an unclear aetiology. Speculation of factors leading to death in these patients is restricted by the very limited availability of laboratory tests. Oliguria and discoloured urine was noted in five of these patients, which could indicate renal toxicity and be an important predictor of severe toxicity or death.

Mechanism of death

Uncoupling of oxidative phosphorylation may be an important component in the cause of death in patients with large chlorophenoxy herbicide exposures. Chlorophenoxy herbicides uncouple oxidative phosphorylation in vitro through an unclear mechanism of action.(16;17)

Uncoupling describes the process whereby oxygen consumption and heat production increase out of proportion to the generation of adenosine triphosphate.(18) It may be caused by extrinsic factors such as chemicals or drugs which disrupt mitochondrial function.(19) Initially this leads to an increase in mitochondrial respiration,(20;21) until the declining levels of ATP are insufficient for essential cellular functions including active transport pumps such as Na-K ATPase. This leads to loss of cellular ionic and volume regulation, and if ATP is not supplied promptly, the effect is irreversible and cell death occurs.(19)

Clinical features consistent with marked uncoupling of oxidative phosphorylation have been reported in cases of human chlorophenoxy herbicide poisoning. Tachypnoea with respiratory alkalosis have been noted in cases of severe poisoning, some of whom died (5;13)(D Roberts and P. Piyasena, unpublished observations), which may be consistent with increased mitochondrial respiration from mild uncoupling. More severe poisoning may be characterised by severe hypoxia, metabolic acidosis, hyperventilation, hyperkalaemia, fever, elevated creatine kinase, tachycardia, generalised muscle rigidity, hypotension, and cardiac asystole.(5;7;22;23) Some of these features were noted in patients who died in our series (Table 3, patients 3,4,7, and perhaps 8). However, these data were not in the dataset collected prospectively under the protocol.

Correlation between plasma pesticide concentrations and toxicity

The relationship between plasma chlorophenoxy herbicide level and toxicity is not clear. A depressed level of consciousness has been reported with plasma chlorophenoxy concentrations from 80 mg/L to over mg/L,(24) while concentrations more than 500 mg/L may be associated with severe toxicity.(25) But death occurred in two patients with a blood MCPA level of 180 mg/L and 230 mg/L.(11;13) A patient survived severe MCPA poisoning (hypotension and limb myotonia) despite a blood concentration of 546 mg/L two hours after ingestion. The muscle effects resolved after a number of days when the MCPA level was less than 100mg/L.(9)

The discordance between admission MCPA levels and the peak toxicity observed during hospital admission is noted in Figure 3. In contrast, concentration dependent uncoupling of oxidative phosphorylation has been demonstrated with rat mitochondria in vitro.(16) The poor correlation between clinical toxicity and plasma concentrations may reflect poor correlation between plasma (measured) and intracellular (mitochondrial) concentrations. Mechanisms which alter the distribution of MCPA between these two spaces includes MCPA-induced damage to cell membranes which increases penetration of the herbicide through cell membranes.(5;26-30) In rats, high plasma concentrations of MCPA damage cell membranes and induce toxicity, but the correlation between plasma levels, membrane damage and toxicity is poor.(29;30)

Another potential mechanism is variation in blood pH, which alters protein binding and tissue distribution. Acidosis increases the proportion of non-ionised MCPA (pKa 3.07), which is lipophilic, and therefore readily crosses the cell membrane, increasing the intracellular concentration. This has been observed in in vitro studies and is similar to that observed for aspirin. (23;31) Similarly, an alkaline plasma pH is expected to decrease tissue binding and increase plasma levels.(25;32;33) Therefore, the distribution of MCPA between plasma and intracellular spaces is likely to vary with plasma pH.

Routine measurement of plasma MCPA levels in patients with intentional self poisoning does not appear useful. Instead, quantification of markers of intracellular toxicity, such as CK from skeletal muscle, may be useful to monitor the effects of MCPA poisoning. Of the patients who had serial blood samples, only two patients (Cases B and C) had significant toxicity (Table 4, Figure 4). Myalgias were reported by these two patients, particularly in Case C where the CK was on admission and increased until discharge. In most patients, peak CK activity was delayed (Figure 4).

Management of patients with MCPA poisoning

All patients should receive routine resuscitation and supportive care. There is insufficient evidence to describe other specific managements of acute MCPA poisoning. It would be reasonable to correct acidosis based on the likely adverse effect of acidosis on MCPA distribution kinetics leading to increased intracellular concentrations.

All significant poisonings (ie symptomatic oral ingestions) should be treated cautiously, preferably in an ICU or other unit with continuous monitoring for 24 to 48 hours. In our series, initial mild toxicity as indicated by normal vital signs and level of consciousness on admission did not preclude subsequent severe toxicity and death. However, we observed that no patients who were completely asymptomatic for the first six hours went on to a fatal outcome. Late deterioration in a reasonably well patient has also been reported with poisoning with MCPP (a related chlorophenoxy herbicide). In this case severe metabolic derangement and death occurred in a patient who was fully conscious prior to an asystolic arrest.(7)

Our data suggest measurement of plasma MCPA confirms exposure but does not provide further information on the severity of poisoning. Confirmation of MCPA exposure may not be necessary in patients presenting with intentional MCPA self-poisoning given that the history was accurate in 88% of patients in our series. We had insufficient data to suggest whether electrolytes, blood gases or CK may be better indicators of the severity of poisoning.

An adequate urine output (>1mL/kg) should be ensured in all patients. Urinary alkalinisation (pH >7.5) and an adequate urine output limits reabsorption and promotes renal excretion.(23;33-35) It might be reasonable to consider these measures in patients who are symptomatic, particularly if they have signs of toxicity. A randomised controlled trial is required to determine the efficacy of this treatment.

Patients with severe toxicity require prompt and effective plasma and urinary alkalinisation, and possibly haemodialysis if facilities are available.(5;35) While there is limited information to support alkalinisation, it is rarely associated with adverse effects when administered carefully and with close observation.(35;36) Signs consistent with uncoupling of oxidative phosphorylation are likely to be associated with a poor outcome.

A better description of the clinical, biochemical, histopathological and histochemical features of severe toxicity and death is required to confirm the mechanism of death in patients. This may help to guide clinical management.

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Conclusion

We found that toxicity from MCPA poisoning is low compared to some other agrochemicals, with 85% of patients having minimal clinical toxicity; but toxicity is still significant with a mortality of 4.4%. Further research is required to identify early markers of poor outcome and to determine if alkalinisation or other interventions can improve the prognosis in this group.

Acknowledgements

Study doctors who assisted with data and sample collection, and hospital doctors for their cooperation with the study. Manel Abeyawardene, Vincent Alberts Brock Jones, and in particular Mary Hodge from Queensland Health Pathology and Scientific Services for assistance with analyses. The Medical Superintendent of Anuradhapura General Hospital for permission to access medical records, and Medical Records staff who located these records. Jason and Alison Roberts for assistance with locating some of the older references.

Reference List

• 1.Eddleston M, Phillips MR. Self poisoning with pesticides. Br.Med.J. ;328:42'4. doi: 10./bmj.328..42. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 2.World Health Organization, editor. World Health Report . World Health Organization; Geneva: . Annex Table 2 Deaths by cause, sex and mortality stratum in WHO Regions, estimates for . [Google Scholar]
• 3.Roberts D, Karunarathna A, Buckley NA, Manuweera G, Sheriff MHR, Eddleston M. Influence of Pesticide Regulation and Integrated Pest Management on Acute Poisoning Deaths in Sri Lanka. Bulletin of the World Health Organization. ;81(10):708'17. [PMC free article] [PubMed] [Google Scholar]
• 4.Donaldson D, Kiely T, Grube A. Pesticides industry sales and usage - and market estimates. Environmental Protection Agency; Washington: . [Google Scholar]
• 5.Bradberry SM, Watt BE, Proudfoot AT, Vale JA. Mechanisms of toxicity, clinical features, and management of acute chlorophenoxy herbicide poisoning: a review. J.Toxicol.Clin.Toxicol. ;38(2):111'22. doi: 10./clt-. [DOI] [PubMed] [Google Scholar]
• 6.Aromataris EC, Astill D.St.J., Rychkov GY, Bryant SH, Bretag AH, Roberts ML. Modulation of the gating of ClC-1 by S-(7) 2-(4-chlorophenoxy) propionic acid. Br.J.Pharmacol. ;126:'82. doi: 10./sj.bjp.. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 7.Dickey W, McAleer JJA, Callender ME. Delayed sudden death after ingestion of MCPP and ioxynil:an unusual presentation of hormonal weedkiller intoxication. Postgrad Med J. ;64:681'2. doi: 10./pgmj.64.755.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 8.Liantonio A, Accardi A, Carbonara G, Fracchiolla G, Loiodice F, Tortorella P, et al. Molecular requisites for drug binding to muscle CLC-1 and renal CLC-K channel revealed by the use of phenoxy-alkyl derivatives of 2-(p-chlorophenoxy)propionic acid. Molecular Pharmacology. ;62(2):265'71. doi: 10./mol.62.2.265. [DOI] [PubMed] [Google Scholar]
• 9.Schmoldt A, Iwersen S, Schluter W. Massive ingestion of the herbicide 2-methyl-4-chlorophenoxyacetic acid. J.Toxicol.Clin.Toxicol. ;35(4):405'8. doi: 10./. [DOI] [PubMed] [Google Scholar]
• 10.Geldmacher von- Mallinckrodt M, Lautenbach L. Zwei todliche Vergiftungen (Suicid) mit chlorierten Phenoxyessigsauren(2,4-D und MCPA) [2 cases of fatal poisoning (suicide) with chlorinated phenoxyacetic acids (2,4-D and MCPA)] Arch Toxikol. ;21:261'78. [PubMed] [Google Scholar]
• 11.Johnson HR, Koumides O. A further case of MCPA poisoning. Br.Med.J. :629'30. doi: 10./bmj.2..629. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 12.Jones DIR, Knight AG, Smith AJ. Attempted suicide with herbicide containing MCPA. Arch Environ Health. ;14:363'6. doi: 10./... [DOI] [PubMed] [Google Scholar]
• 13.Popham RD, Davies DM. A case of M.C.P.A. poisoning. BMJ. ;1:677'8. doi: 10./bmj.1..677-a. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 14.Coyne BA. Chemically induced or inherited myotonia? Emerg.Med. ;5:149'244. [Google Scholar]
• 15.Senarathna L, Mohammad F, Eddleston M. Correlation of human toxicity case fatality ratio (CFR) for pesticides with animal LD50 [abstract]. The Asia Pacific Association of Medical Toxicology 4th International Conference, Philippines.. p. 26. [Google Scholar]
• 16.Zychlinski L, Zolnierowicz S. Comparison of uncoupling activities of chlorophenoxyherbicides in rat liver mitochondria. Toxicol.Lett. ;52:25'34. doi: 10./-(90)-f. [DOI] [PubMed] [Google Scholar]
• 17.Brody TM. Effect of certain plant growth substances on oxidative phosphorylation in rat liver mitochondria. Proc.Soc.Exp.Biol.Med. ;80:533'6. doi: 10./-80-. [DOI] [PubMed] [Google Scholar]
• 18.Kadenbach B. Intrinsic and extrinsic uncoupling of oxidative phosphorylation. Biochimica et Biophysica Acta. ;:77'94. doi: 10./s-(03)-6. [DOI] [PubMed] [Google Scholar]
• 19.Gregus Z, Klaassen CD. Mechanisms of toxicity. In: Klaassen CD, editor. Casarett and Doull's Toxicology - The basic science of poisons. 5th ed. McGraw-Hill; New York: . pp. 35'74. [Google Scholar]
• 20.Brody TM. Action of sodium salicylate and related compounds on tissue metabolism in vitro. J Pharmacol Exp Ther. ;117(1):39'51. [PubMed] [Google Scholar]
• 21.Chance B, Williams GR. Respiratory enzymes in oxidative phosphorylation. I. Kinetics of oxygen utilization. J Biol Chem. ;217:383'93. [PubMed] [Google Scholar]
• 22.O'Reilly JF. Prolonged coma anddelayed peripheral neuropathy after ingestion of phenoxyacetic acid weedkillers. Postgrad.Med.J. ;60:76'7. doi: 10./pgmj.60.699.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 23.Yip L. Salicylates. In: Dart RC, editor. Medical Toxicology. 3 ed. Lippincott Williams Wilkins; Philadelphia: . pp. 739'49. [Google Scholar]
• 24.Chlorophenoxy herbicides . Recognition and management of pesticide poisonings. In: Reigart JR, Roberts JR, editors. 5 ed. United States Environmental Protection Agency; Washington: . pp. 94'9. [Google Scholar]
• 25.Flanagan RJ, Meredith TJ, Ruprah M, Onyon LJ, Liddle A. Alkaline diuresis for acute poisoning with chlorophenoxy herbicides and ioxynil. Lancet. ;335():454'8. doi: 10./-(90)-w. [DOI] [PubMed] [Google Scholar]
• 26.Bukowska B, Goszczynska K, Duda W. Effect of 4-chloro-2-methylphenoxyacetic acid and 2,4-dimethylphenol on human erythrocytes. Pesticide Biochemistry and Physiology. ;77:92'8. [Google Scholar]
• 27.Elo H, Ylitalo P. Substantial increase in levels of chlorophenoxyacetica acids in the CNS of rats as a result of severe intoxication. Acta Pharmacol Et Toxicol. ;41:280'4. doi: 10./j.-..tb.x. [DOI] [PubMed] [Google Scholar]
• 28.Elo HA, Ylitalo P, Kyotilla J, Hervonen H. Increase in the penetration of tracer compounds into the rat brain during 2-methyl-4-chlorophenoxyacetic acid (MCPA) intoxication. Acta Pharmacol Et Toxicol. ;50:104'7. doi: 10./j.-..tb.x. [DOI] [PubMed] [Google Scholar]
• 29.Hervonen H, Elo HA, Ylitalo P. Blood-brain barrier damage by 2-methyl-4-chlorophenoxyacetic acid herbicide in rats. Toxicol.Appl.Pharmacol. ;65:23'31. doi: 10./-008x(82)-1. [DOI] [PubMed] [Google Scholar]
• 30.Elo HA, Ylitalo P. Distribution of 2-methyl-4-chlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid in male rats: Evidence for the involvement of the central nervous system in their toxicity. Toxicol.Appl.Pharmacol. ;51:439'46. doi: 10./-008x(79)-5. [DOI] [PubMed] [Google Scholar]
• 31.Cabral MG, Viegas CA, Teixeira MC, Sa-Correia I. Toxicity of chlorinated phenoxyacetic acid herbicides in the experimental eukaryotic model Saccharomyces cerevisiae: role of pH and of growth phase and size of the yeast cell population. Chemosphere. ;51:47'54. doi: 10./s-(02)-8. [DOI] [PubMed] [Google Scholar]
• 32.Arnold EK, Beasley VR. The pharmacokinetics of chlorinated phenoxy acid herbicides: a literature review. Vet Hum Toxicol. ;31(2):121'5. [PubMed] [Google Scholar]
• 33.Rowland M, Tozer TN. Clinical Pharmacokinetics - Concepts and Applications. 2nded Lea & Febiger; Philadelphia: . [Google Scholar]
• 34.Braunlich H, Bernhardt H, Bernhardt I. Renal handling of 2-Methyl-4-Chlorophenoxyacetic acid (MCPA) in rats. Journal of Applied Toxicology. ;9(4):255'8. doi: 10./jat.. [DOI] [PubMed] [Google Scholar]
• 35.Proudfoot AT, Krenzelok EP, Vale JA. AACT/EAPCCT position paper on urinary alkalinisation. J.Toxicol.Clin.Toxicol. ;42(1):1'26. doi: 10./clt-. [DOI] [PubMed] [Google Scholar]
• 36.Liebelt EL. Sodium bicarbonate. In: Dart EC, et al., editors. Medical Toxicology. 3 ed. Philadelphia; Lippincott Williams Wilkins: . pp. 257'61. [Google Scholar]
• 37.Singh S, Yadav S, Sharma N, Malhotra P, Bambery P. Fatal 2,4-D (ethyl ester) ingestion. JAPI. ;51:609'10. [PubMed] [Google Scholar]
• 38.Bronstein AC. Herbicides. In: Dart EC, editor. Medical Toxicology. 3 ed. Lippincott Williams Wilkins; Philadelphia: . pp. '29. [Google Scholar]
• 39.O'Malley M. Chlorophenoxy herbicides. In: Olsen KM, editor. Poisoning and drug overdose. 4 ed. McGraw-Hill Companies; San Francisco: . pp. 164'5. [Google Scholar]
• 40.Henry J, Wiseman H. Management of poisoning - A handbook for healthcare workers. WHO International Programme on Chemical Safety; Geneva: . [Google Scholar]
• 41.Farm chemicals handbook '99. Meister Publishing Company; Willoughby: . [Google Scholar]
• 42.IPCS . World Health Organization; Geneva: . WHO recommended classification of pesticides by hazard and guidelines to classification -. [Google Scholar]

Background

Cotton (Gossypium hirsutum) is an important economic crop in many places in the world [1]. In the process of cotton growth, often stress by pests and weeds. For a long time, the use of pesticides can reduce the harm of pests and weeds to cotton. However, in the use of pesticides, due to the nonstandard operation, pesticide damage often occurs in cotton, and it may occur at all growth stages of cotton [2]. Pesticide damage often causes cotton leaf deformity, chlorosis, and buds, flowers and young bolls were being prone to drop [3]. In particular, the herbicide damage has a great impact on the reduction of cotton yield. Therefore, plant growth regulators are widely used to alleviate and reduce the impact of herbicide damage on cotton, and are often used as remedial measures to reduce cotton yield loss [4].

2-methyl-4-chlorophenoxy acetic acid-Na (MCPA-Na) is a phenoxyacetic acid selective hormone herbicide [5]. It exposure disrupts the transportation tissue in dicotyledonous plants, interfering with plant growth and development, and thereby, achieving the purpose of weeding [6]. In China, MCPA-Na is widely used to control broad-leaf weeds such as pondweed, meadow pine, and sedge in wheat, rice, sugarcane and flax fields [7'10]. In addition, MCPA-Na is also used for inter-row directional weed control in the cotton fields [11]. However, due to the lack of a protective cover on the application nozzle, drift as well as direct exposure can also cause serious damage to the non target G. hirsutum plants [12]. Reducing the impact of cotton damage and the subsequent losses in yield is therefore essential.

For a long time, herbicide damage has been an important limiting factor of crop yield, especially in relation to inactivated herbicides [13'15]. Field observations show that cotton is extremely sensitive to MCPA-Na. Leaves of cotton plants exposed to this herbicide become pale and brittle as well as wrinkled and thickened with prominent veins, while the overall leaf shape becomes narrow, leaf margins of young leaves curl upwards, becoming cup-shaped, bracts turn red, and buds, flowers, and young bolls were being prone to drop [12]. Due to the inability of cotton to metabolize MCPA-Na, callus-like growths also develop on the lower stem and roots, resulting in the wilting of young buds and tissue necrosis [12]. Moreover, this damage often occurs during important growth periods, such as the seedling, bud, flowering or boll stages. In our previous study, exposure of cotton seedlings to MCPA-Na caused an increase in the soluble protein content and protective enzyme activity [13]. However, whether these modifications occur following MCPA-Na exposure during other growth stages and the subsequent effects on cotton yield remain unknown.

Plant growth regulators play an important role in regulating crop growth and development, improving yield and quality, and enhancing stress resistance [16, 17]. Notably, growth regulators improve the ability of plants to resist drought, salinity, and pests [18'22]. In addition, they are also used to alleviate pesticide damage. For example, brassinosteroids protects maize from amphetamine damage [21], while the combined application of sodium nitrophenolate, choline chloride and inositol reduce pyraclostrobin damage in soybean and maize seedlings [22]. Similarly, gibberellic acid was found to alleviate S-metolachlor damage in rice seedlings [23]. Recent research further suggests that plant growth regulators alleviate damage by improving the activity of protective enzymes [24]. However, their effectiveness in alleviating MCPA-Na toxicity in cotton is yet to be determined. Clarification of the detoxification mechanism of plant growth regulators in MCPA-Na-exposed G. hirsutum is therefore important.

During this study, we examined the effects of MCPA-Na on physiometabolic activities and yield traits in cotton following herbicide exposure at the seedling, budding, flowering and boll stages. Four plant growth regulators (24-epibrassinolide, GA3, phthalanilic acid and seaweed fertilizer) were used following MCPA-Na exposure, which could promote cotton plants growth. The protective effects of different combinations of plant growth regulators on MCPA-Na phytotoxicity were then examined at each growth stage. The results of this study provide an understanding of the underlying mechanism of MCPA-Na toxicity in G. hirsutum, supporting measures aimed at the use of plant growth regulators to alleviate damage, which has important practical significance for cotton producers. And the study provides a case for the solution of herbicide damage in other crops.

Materials and methods

Experimental field and plants

The field experiment was conducted at Shihezi University Educational Test Site, Xinjiang, China (86°E, 44°N). The soil was loam, containing 7.51'mg·kg''1 organic carbon, 1.32'g·kg''1 total nitrogen, 185'mg·kg''1 available potassium and 7.12'mg·kg''1 available phosphorus. The land has been growing cotton for more than 5'years. The experiment was conducted from to .

The cotton variety used was Xinluzao 12 (provided by Institute of Cotton Research, Chinese Academy of Agricultural Sciences), and the seeds were treated with imidacloprid seed coating agent (Gaucho® Bayer Crop Science, 6'mL'kg-1 seeds). Seeding was carried out under mulch film with width of 1.5'm. Row were spaced at (30'+'50'+'30) cm, the cotton plants were spaced above 10'cm, and with a planting density of 150,000 plants/hm2.

Experimental field management

Before sowing, (NH4)3PO4 @ 175'kg hm-2 and urea @ 260'kg hm-2 were applied as basal fertilizer. The seeds were sown on 15 April, and 25 April, . Irrigation was carried out four times throughout the growing period on (28 June, 18 July, 8 August, and 24 August) and (15 June, 7 July, 1 August, and 19 August), respectively. Drip irrigation was also carried out using and m3 hm-2 water on and , respectively. During the cotton growth period (June'August), 525'kg hm-2 urea, and 450'kg hm-2 KH2PO4·NH4H2PO4 were also fertilization through drip irrigation. Chemical capping was carried out on (10 July and 25 July) and (3 July and 20 July) using 250'300'g hm-2 mepiquat chloride (Pix® HC, Basf), respectively. Pest and disease control were carried out according to the standard management practices, mainly to control cotton aphids, thrips and spider mites.

MPCA-Na and plant growth regulators

The following plant growth regulators were used: 13% MPCA-Na aqueous solution (Songrun Pharmaceutical Factory, Jilin, China), 0.% 24-epibrassinolide aqueous solution (Xinchaoyang Crop Science Co., Ltd., Chengdu, China), 75% Gibberellin (GA3) crystal powder (Tongrui Biotechnology Co., Ltd., Shanghai, China), seaweed fertilizer (Xinhefeng Agrochemical Information Co., Ltd., Beijing, China), and 20% Phthalanilic acid soluble concentrate (Sunger Bioscience Co., Ltd., Shanxi, China).

MCPA-Na application

According to the recommended dose of 13% MCPA-Na, concentrations of 0, 8.125, 16.25, 32.5, 65, and 130'g L-1 (500'kg water hm-2) were configured for use in this study. In the preparation of MCPA-Na with different concentrations, 8.125, 16.25, 32.5, 65, and 130'g MCPA-Na were weighed with electronic balance, respectively. Then, poured into a bucket containing 1'L of water, and stirred evenly with a glass rod. The prepared of MCPA-Na was poured into a 3WBD-20'L back-loaded electric sprayer. The preparation was repeated according to the above steps until the sprayer was filled with the MCPA-Na.

The experiment followed a completely randomized block design, with each plot measuring an area of 100 m2 (10'm'×'10'm), which contained nine rows in the plot. A 3WBD-20'L back-loaded electric sprayer was used to evenly apply MCPA-Na at the seedling (growth of the fourth true leaf), budding, flowering and boll stages. Each treatment was repeated three times. Contents of chlorophyll, soluble protein and MDA, and activities of SOD, CAT and POD in the cotton leaves were then measured on days 1, 4, and 7 after treatment. Plant height, boll number and the single boll weight were also measured on 15 October and 10 October (harvest period) to determine the effect on yield.

Application of plant growth regulators following MCPA-Na exposure

A complete randomized block design was also used to study the effects of the plant growth regulators, with each plot measuring 100 m2. MCPA-Na at the maximum concentration of 130'g L-1 was applied using the 3WBD-20'L back-loaded electric sprayer at the seedling, budding, flowering and boll stages. Two days after spraying MCPA-Na, different combinations of plant growth regulators were then applied (see Table 1 for each treatment combination). T0 (control), T1 (24-epibrassinolide), T2 (GA3'+'seaweed fertilizer), T3 (24-epibrassinolide + seaweed fertilizer) and T4 (phthalanilic acid + seaweed fertilizer), T5 (GA3), T6 (24-epibrassinolide + GA3'+'phthalanilic acid + seaweed fertilizer), T7 (phthalanilic acid), T8 (24-epibrassinolide + GA3'+'phthalanilic acid).

Table 1.

Gibberellin Brassinosteroids Phthalanilic acid Seaweed fertilizer T0 ' ' ' ' T1 ' + ' ' T2 + ' ' + T3 ' + ' + T4 ' ' + + T5 + ' ' ' T6 + + + + T7 ' ' + ' T8 + + + '

0.% 24-epibrassinolide, 75% gibberellin (GA3), 20% phthalanilic acid and seaweed fertilizer were applied at dosages of 0.25'mL L-1, 0.01'g L-1, 0.5'g L-1, and 3.5'mL L-1 (500'kg water hm-2), all of which represent the recommended concentrations of each component. Preparation of T1: 0.25'mL 24-epibrassinolide was poured into bucket filled with 1'L water. Preparation of T2: 0.01'g GA3 and 3.5'mL seaweed fertilizer were poured into bucket filled with 1'L water. Preparation of T3: 0.25'mL 24-epibrassinolide and 3.5'mL seaweed fertilizer were pour into bucket filled with 1'L water. Preparation of T4: 0.5'g phenylamino acid and 3.5'mL seaweed fertilizer were pour into bucket filled with 1'L water. Preparation of T5: 0.01'g GA3 was poured into bucket filled with 1'L water. Preparation of T6: 0.25'mL 24-epibrassinolide, 0.01'g GA3, 0.5'g phthalanilic acid and 3.5'mL seaweed fertilizer were poured into bucket filled with 1'L water. Preparation of T7: 0.5'g phenylamino acid was poured into bucket filled with 1'L water. Preparation of T8: 0.25'mL 24-epibrassinolide, 0.01'g GA3 and 0.5'g phthalanilic acid were poured into bucket filled with 1'L water. Each treatment was repeated three times. Contents of chlorophyll, soluble protein and MDA, and activities of SOD, CAT and POD in the cotton leaves were then measured as above on days 1, 4 and 7 after treatment. Plant height, boll number and the single boll weight were also measured on 15 October and 10 October (harvest period) to determine the effect on yield.

Analysis of chlorophyll, soluble protein, MDA and protective enzyme activity in the cotton leaves

Chlorophyll content was measured using the acetone ethanol method [25], soluble protein was determined using the G-250 dye colorimetric method [26], and the MDA content was determined based on the thiobarbituric acid method [27].CAT, POD and SOD activity were determined using the guaiacol method [28], UV absorption method [29] and riboflavin-NBT method [30], respectively.

Analysis of plant height, boll number and the single boll weight

On 15 October and 10 October , 20 cotton plants were randomly selected from each treatment for analysis of plant height, boll number and the single boll weight. Plant height was determined as the length from the stem base to the growing point. The number of cotton bolls per plant, and the single boll weight was determined as the lint weight of a single cotton boll. Each treatment was repeated three times then mean values were obtained.

Statistical analysis

Microsoft Excel was used for data processing and plotting. SPSS 20.0 data processing software was used for all statistical analyses, giving mean values and standard errors. The LSD test was used to test for significance between the differences in means.

Results

Effects of MCPA-Na exposure and application of plant growth regulators at the seedling stage

MPCA-Na exposure at the seedling stage caused a decrease in chlorophyll, and increases in contents of soluble protein and MDA, and activity of all protective enzymes. Plant height, boll number and the single boll weight all decreased with increasing MPCA-Na (Fig. 1). Meanwhile, with time, the contents of chlorophyll and soluble protein decreased, contents of MDA and activities of SOD and POD increased, and activity of CAT decreased (Fig. S1).

Compared with the control, treatment with a high concentration of MPCA-Na (130'g L-1) caused decreases in the chlorophyll content of 30.72, 29.93, and 41.86% (p'<'0.05) on days 1, 4 and 7 after treatment, respectively (Fig. 1A). Meanwhile, the soluble protein content increased by 34.62, 74.99 and 68.97%, while MDA level increased by 3.90, 5.43, and 34.95%, respectively (p'<'0.05, Fig. 1B & C). The SOD activity decreased by 34.30%on day 1 after treatment then increased by 20.90 and 25.42% on days 4 and 7, respectively (p'<'0.05, Fig. 1D). Meanwhile, POD activity increased by 76.28, 128.85, and 319.01%, while CAT activity increased by 44.68, 79.31, and 45.78% (p'>'0.05), respectively (p'>'0.05, Fig. 1E & F). Compared with the control, plant height decreased by 42.08%, the number of bolls decreased by 75.33%, and the single boll weight decreased by 46.42% following treatment with 130'g L-1 MPCA-Na (p'<'0.05, Fig. 1G, H & I).

Application of the plant growth regulators caused a decrease in the contents of chlorophyll, soluble protein and MDA, and had a positive effect on protective enzyme activity, and reducing cotton yield losses (Fig. 2). With time, the chlorophyll and soluble protein contents increased then decreased, while the MDA content showed a gradual increase, SOD and POD activity decreased, and CAT activity increased then decreased (Fig. S2).

On days 1 and 4, the chlorophyll content was significantly lower under T6 than T1, T2, T3 and T4 (Fig. 2A). Meanwhile, on days 1, 4 and 7, the soluble protein content was significantly lower under T3 and T4 than T1 (Fig. 2B). The MDA content was significantly lower under T8 than T1 (Fig. 2C), while on day 7, SOD activity was significantly higher under T4 and T6 than all other treatment groups (Fig. 2D). On day 4, highest POD activity was observed under T4 at 763.33'U g''1  min-1 (Fig. 2E), while highest CAT activity was observed under T8 at 666.67'U g''1  min-1 (Fig. 2F). Plant height was highest under T5 (Fig. 2G), while boll number and single boll weight were highest under T4 (Fig. 2H & I). It should be treated with phthalanilic acid + seaweed fertilizer at seedling stage to mitigate stress of MCPA-Na damage.

Effects of MCPA-Na exposure and application of plant growth regulators at the budding stage

After MPCA-Na exposure at the budding stage, the content of chlorophyll decreased, while contents of soluble protein and MDA, and activity of the protective enzymes increased. Plant height, boll number and the single boll weight decreased with increasing MPCA-Na concentration (Fig. 3). With time, contents of chlorophyll and soluble protein gradually decreased, while the MDA content, and SOD and POD activities increased following treatment with 130'g L-1 MPCA-Na. CAT activity first increased then decreased (Fig. S3).

Compared with the control, the chlorophyll content decreased by 5.42, 23.68, and 38.07% on days 1, 4 and 7 after treatment with 130'g L-1 MPCA-Na, respectively (p'<'0.05, Fig. 3A), while the soluble protein content increased by 9.75 and 2.53% on days 1 and 4 then decreased by 41.52% on day 7 (p'<'0.05, Fig. 3B). The MDA content increased by 21.49, 92.10, and 164.19%, respectively (p'>'0.05, Fig. 3C), while SOD activity decreased by 0.21% on day 1 then increased by 11.50 and 3.30% on days 4 and 7 (p'<'0.05, Fig. 3D). POD activity increased by 73.32, 75.01, and 99.56%,while CAT activity increased by 22.32 and 25.95% on days 1 and 4 then decreased by 6.25% on day 7 (p'<'0.05, Fig. 3E & F). Compared with the control, plant height decreased by 54.95%, the number of bolls decreased by 79.50%, and the single boll weight decreased by 36.31% following treatment with 130'g L-1 MPCA-Na (p'<'0.05, Fig. 3G, H & I).

Application of plant growth regulators increased the chlorophyll and soluble protein content, decreased the MDA content, and improved the protective enzyme activity, and reducing cotton yield losses (Fig. 4). With time, the chlorophyll, soluble protein, and MDA contents decreased along with POD activity, and improved SOD and CAT activity (Fig. S4).

On days 1 and 4, the chlorophyll content was significantly higher under T2, T3, T4 and T8 than T6 (Fig. 4A). On day 7, the soluble protein content was highest under T2 at 23.0'mg g-1 (Fig. 4B), while on days 1, 4 and 7, the MDA content was significantly lower under T4 and T5 than under T1 and T2 (Fig. 4C). On day 1, SOD activity was highest under T8 at 48.75'U g''1  min-1 (Fig. 4D), while POD activity was highest under T1 at 535.35'U g''1  min-1 (Fig. 4E). On day 7, CAT activity was highest under T2 at 341.41'U g''1  min-1 (Fig. 4F). Plant height was highest under T6 (Fig. 4G), while the number of cotton bolls was highest under T3 (Fig. 4H), and the single boll weight was highest under T7 (Fig. 4I). It should be treated with 24-epibrassinolide + seaweed fertilizer at buddling stage to mitigate stress of MCPA-Na damage.

Effects of MCPA-Na exposure and application of plant growth regulators at the flowering and boll stages

MPCA-Na exposure decreased the chlorophyll content, increased the soluble protein content, MDA content and protective enzyme activity, had little effect on plant height and boll weight, and reduced the boll number at both the flowering and boll stages (Fig. 5). Meanwhile, with time, the chlorophyll content decreased, while the soluble protein content, MDA content and protective enzyme activity increased (Fig. S5).

Compared with the control, the chlorophyll content under 130'g L-1 MPCA-Na decreased by 37.37, 23.39, and 45.78% on days 1, 4 and 7, respectively (p'<'0.05, Fig. 5A), while the soluble protein content decreased by 7.87% on day 4 then increased by 7.25 and 20.31% on days 1 and 7, respectively (p'<'0.05, Fig. 5B). The MDA content through different treatments increased by 8.74, 45.15 and 34.34%, respectively at p'<'0.05 (Fig. 5C). Meanwhile, SOD activity increased by 48.91, 119.85, and 137.03%, POD activity increased by 45.71, 54.69, and 93.62%, and CAT activity increased by 28.44, 31.81, and 53.89%, respectively (p'<'0.05, Fig. 5D, E & F). Plant height increased by 2.82%, boll number decreased by 48.15%, and the single boll weight decreased by 5.38% (p'>'0.05, Fig. 5G, H & I).

Application of the plant growth regulators increased the protective enzyme activity, reduced the MDA content, and stabilized plant height and the single boll weight (Fig. 6). With time, the chlorophyll and soluble protein content increased then decreased (except under T7), while the MDA content gradually increased, SOD (except under T1) and POD activity decreased, and CAT activity gradually increased under T1 only (Fig. S6).

On day 4, the chlorophyll and soluble protein contents were highest under T2 at 1.84 and 4.39'mg g-1, respectively (Fig. 6A & B), while the MDA content was significantly lower under T5 than T1 (Fig. 6C). CAT activity was highest under T3 at .33'U g''1  min-1 (Fig. 6F), while on day 1, SOD activity was highest under T2 at 808.00'U g''1  min-1 (Fig. 6D) and POD activity was highest under T7 at 664.83'U g''1  min-1 (Fig. 6E). Plant height was highest under T5 (Fig. 6G), the number of cotton bolls was highest under T1 (Fig. 6H), and the single boll weight was highest under T2 (Fig. 6I). It should be treated with GA3'+'seaweed fertilizer at flowering and boll stage to mitigate stress of MCPA-Na damage.

Discussion

Most herbicides have injurious effects on crop growth, causing leaves to shrink, inhibiting plant height, and causing flowers and fruit to drop, all of which cause serious reductions in crop yield [31, 32]. After exposure, crops tend to regulate their physiological and metabolic functions in order to convert herbicides into harmless compounds [33]. However, not all herbicides can be metabolized. For example, MCPA-Na is not metabolized by cotton [6], therefore such plants need to actively regulate their physiological metabolism in order to reduce subsequent damage. The regulatory mechanisms underlying protective enzyme responses to damage have been confirmed in a number of plants [34]. For example, 2, 4-D was found to cause an increase in protective enzyme activity in wheat and rice [35, 36], while MCPA causes an increase in protective enzyme activity in millet [37], and MCPA-Na induces an increase in bloom-forming cyanobacteria protective enzyme activity [38]. In this study, MCPA-Na exposure at different growth stages caused increases in the soluble protein content and protective enzyme activity, and decreases in the chlorophyll content. These findings suggest that cotton does not minimize MCPA-Na damage by enhancing photosynthesis, but rather by increasing basal metabolism and protective enzyme activity.

The growth period of plants determines their sensitivity to herbicides, as shown by the sensitivity of cotton, cucumber and watermelon to 2, 4-D exposure [39'41]. In this study, resistance of cotton to MCPA-Na at different growth stages was also found to differ. For example, at the seedling stage, MDA began to accumulate on day 7 after exposure, while at the budding stage, accumulation began on day 4. Moreover, at the flowering and boll stages, the MDA content was lower than the control on days 1, 4 and 7 at MCPA-Na concentrations of 8.125 and 16.25'g L-1 MCPA-Na. Meanwhile, plant height and the single boll weight were less affected by MCPA-Na damage at the flowering and boll stages, but together with boll number were significantly affected at the seedling and budding stages. These findings suggest that resistance of cotton to MCPA-Na is stronger at the flowering and boll stages than at the seedling and budding stages. In other words, the vegetative growth phase is more sensitive to MCPA-Na toxicity.

The limited defense capacity of plants is unable to provide sustainable or high-level metabolic responses during exposure to herbicides that are slowly metabolized or even unable to be metabolized in plants [42, 43]. Moreover, with increased exposure, plants are more prone to serious damage or even death [44, 45]. When multiple reactive oxygen species cannot be eliminated by the antioxidant enzyme system, oxidative stress and lipidization of the cell membrane are induced, resulting in an accumulation of MDA in vivo [46, 47]. In this study, the increased exposure to MCPA-Na, notably on day 7, caused an increase in MDA accumulation, which was not eliminated by normal metabolism. This finding suggests that the defense responses of cotton are unable to cope with prolonged MCPA-Na damage, highlighting the need for additional measures to enhance the defense ability and reduce damage.

Plant growth regulators are often used to alleviate herbicide damage [48]. For example, brassinosteroids were found to protect maize from amphetamine damage [21], while gibberellin can alleviate S-metolachlor damage in rice seedlings [23]. In general, plant growth regulators help plants increase their metabolic levels, including antioxidant capacity and related gene expression [49'53], and reducing herbicide damage [54, 55]. In this study, combined application of plant growth regulators improved physiological metabolism in cotton exposed to MCPA-Na, increasing the chlorophyll content and reducing the MDA content. It is worth noting that specific plant growth regulators should be used in different growth stages of cotton, because the demand for plant growth regulators is different in different growth stages of cotton [52, 53], and the resistance of cotton to herbicide damage is also different. From a physiological point of view, the application of phthalanilic acid + seaweed fertilizer (T4) alleviated MCPA-Na damage at the seedling stage, the application of 24-epibrassinolide + seaweed fertilizer (T3) alleviated MCPA-Na damage at the budding stage, while at the flowering and boll stage, the application of GA3'+'seaweed fertilizer (T2) is recommended.

There are antagonistic or synergistic effects between different kinds of plant growth regulators. It is necessary to use them reasonably according to the characteristics of their effects to reduce herbicide damage. 24-epibrassinolide can effectively improve the chlorophyll content and photosynthetic efficiency of plants [56, 57], protect flowers and fruits [58], and reduce the occurrence of drug damage [59]. GA3 can promote plant flowering [60], increase fruit setting rate and yield [61]. Phthalanilic acid can promote the transport of nutrients to growth points such as buds, enhance plant cell viability, promote chlorophyll synthesis, and have a synergistic effect with auxin [62, 63]. As a natural seaweed extract, seaweed fertilizer has the dual functions of plant regulation and nutrition supplementation [64], promoting root development and improving absorption and utilization, and can also protect flowers and fruits and improve fruit yield and quality [65, 66]. Therefore, in this study, we used the characteristics of different kinds of plant growth regulators, and combined these plant growth regulators in different damage stages of cotton. In the seedling stage, after being damaged by MCPA-Na, the combination of phthalanilic acid and seaweed fertilizer can synergistically promote the vegetative growth of cotton, promote the synthesis of chlorophyll and root development, improve the absorption and utilization of nutrients and antioxidant capacity, and can better resist MCPA-Na. In the budding stage, the synergistic effect of 24-epibrassinolide and seaweed fertilizer can protect cotton buds, improve photosynthesis and antioxidant capacity, and promote reproductive growth. In the flowering and boll stage, the synergistic effect of GA3 and seaweed fertilizer can promote the boll setting of cotton and increase the yield. We suggest that when using plant growth regulators to alleviate the phytotoxicity of cotton, it should be used in a scientific combination according to the characteristics of cotton growth stage and plant growth regulators, so as to avoid antagonism and secondary phytotoxicity when using growth regulators.

Under a fixed planting density, cotton yield is determined by the number of bolls and the single boll weight [67]. Herbicide damage tends to reduce both traits, thereby reducing yield [68'70]. A recent study also found that cotton yield is differentially affected by different herbicides at different growth stages [39]. For example, 2, 4-D was found to have differential effects on the single boll weight and boll number at different growth stages [39]. In this study, MCPA-Na affected cotton yield by reducing the boll number and single boll weight at the seedling and budding stages, and by reducing the boll number at the flowering and boll stage. Compared with the seedling and budding stage, the effect on yield is relatively small at the flowering and boll stage that exposure by MCPA-Na. Meanwhile, the application of plant growth regulators caused an increase in the single boll weight and boll number following MCPA-Na exposure.

In terms of yield, the findings of this study suggest that cotton exposed to MCPA-Na at the seedling stage should be treated with phthalanilic acid + seaweed fertilizer, while plants exposed at the budding stage should be treated with 24-epibrassinolide + seaweed fertilizer, and those exposed at the flowering and boll stage should be treated with GA3'+'seaweed. However, despite these findings, the effect of the plant growth regulators on cotton damaged at the flowering and boll stage was less than satisfactory, causing branches and leaves lush and boll opening difficulties at later stages. Therefore, for cotton exposed at these later stages, we suggest a low dosage of plant growth regulators to avoid shoot elongation and reduce the impact on yield.

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