review of nioh study of endosulfan in kerala, india

Review of Scientific Methods and Findings for:Final Report of The Investigation of UnusualIllnesses Allegedly Produced by EndosulfanExposure in Padre Village of Kasargod District (N.Kerala)

Robert P. DeMott1, Thomas D. Gauthier1, Diane J. Mundt2

1 - ENVIRON International Corporation, Tampa, Florida, USA2 - ENVIRON International Corporation, Boston, Massachusetts, USA

Corresponding Author

Robert P. DeMott, Ph.D., Principal ToxicologistENVIRON International10150 Highland Manor Dr., Suite 440Tampa, FL 33610 [email protected]

Submitted -- March 27, 2012

Scientific Review –NIOH Endosulfan Study

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Executive Summary

This report evaluates the scientific methods, conduct, interpretations and conclusion of a report

titled “Final Report of The Investigation of Unusual Illnesses Allegedly Produced by Endosulfan

Exposure in Padre Village of Kasargod District (N. Kerala)”conducted by the Indian National

Institute of Occupational Health (NIOH). This is a project report of a study regarding

environmental and human health conditions in an area near cashew plantations where there

has been a history of aerial application of the pesticide endosulfan. The report covers

environmental sampling of soil, water, sediment and other materials. It also includes a survey

used to obtain information about human health conditions and follow-up testing involving

physical examinations and blood sampling for endosulfan and hormonal analyses. The report

reaches broad and strong conclusions that human health conditions grouped under three

headings (neurobehavioral disorders, male reproductive system abnormalities, and congenital

malformations in females) are more prevalent in Padre village (the “study”population), near the

sprayed plantations, than in a reference village approximately 25 km distant where this

endosulfan application method was reportedly not used. The report goes so far as to conclude

that endosulfan is the “most probable”cause of the conditions documented in the report.

The evaluation of this report covers the study design, the analytical chemistry methods and the

relevance of the findings to endosulfan effects. First, a necessary limitation of the study design

employed to evaluate human health conditions, termed a survey study, is that it cannot reach

conclusions regarding the causes of effects noted. This method is generally accepted as

suitable solely for preliminary hypothesis generation. Based solely on the study design

selected, ascribing endosulfan as the most probable cause of the conditions recorded is not a

scientifically valid conclusion, even; without considering the quality of its conduct and findings.

When we do consider the analyses conducted and the interpretations, however, serious

limitations and inadequacies emerge. There were serious omissions and errors in the

computation and presentation of the analytical results for endosulfan in environmental samples.

In fact, there was not even confirmation that the measurements made were actually endosulfan.

Lack of confirmation, calibration and quality assurance/quality control information are such that

these results would commonly be rejected by environmental regulatory agencies and would not

be considered reliable for characterizing human health risks for any type of regulatory or judicial

action.

The survey method as conducted to obtain information regarding human health conditions does

not conform to the generally accepted requirements for epidemiological studies and is

insufficient for reaching a conclusion that endosulfan effects are observed in Padre village.

Substantial sources of potential bias were not prevented or considered in the analysis, and then

were not discussed in the report as possible sources of uncertainty and limitations. The

comparisons were frequently simplistic and, in some cases, presented in ways that served to

mask or overlook information that would have weakened the conclusion of adverse effects

occurring more prevalently in Padre village. The epidemiological results obtained and the


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analyses conducted also to not meet the standards commonly required for studies used to

support regulatory or judicial action.

The effects reported to be linked to endosulfan are not biologically plausible as toxicological

responses to the low, background levels of endosulfan found. The congenital conditions

highlighted for male reproductive effects, in fact, are not even found in excess among the boys

from Padre village. And, the measurements characterized to reflect neurobehavioral disorders

are not generally accepted as direct markers of such effects. The interpretations stretch highly

uncertain and highly subjective tests into such serious terms as “disorders,”“abnormalities,”and

“malformations.”The results do not support the presence of elevated rates of such conditions in

Padre village. The analyses and interpretations do not meet generally accepted scientific

standards for establishing chemical exposures as an explanation for purported health effects.

Finally, there were significant and serious discrepancies in some cases between the raw data

obtained through a Right to Information request, the presentations made in the report, and the

subsequent presentations made in a journal article published from the study. Sample results

were excluded, transcription errors were made and numbers of subjects were changed in ways

that served to make the conclusions of the report and the journal article in particular stronger. In

conclusion, the NIOH Report cannot be used to draw a causal connection meeting generally

accepted scientific standards between endosulfan exposure and various reported symptoms

and outcomes because of the limitations of the design, the uncertainties and inadequacies of

the analyses, and the lack of concordance between the reported findings and actual adverse

health conditions.


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1 Introduction

This report provides a critical review of the study prepared by the National Institute of

Occupational Health (NIOH), Indian Council of Medical Research, titled “Final Report of The

Investigation of Unusual Illnesses Allegedly Produced by Endosulfan Exposure in Padre Village

of Kasargod District (N. Kerala)”(hereafter referred to as the “NIOH Report”).

1.1 Background

The NIOH Report was prepared in response to several reports in the press of unusual diseases

in residents of small villages in the Kasargod district of Northern Kerala. The villages are

located below hilltop cashew plantations that have been treated for control of tea mosquitoes by

aerially spraying with endosulfan insecticide two to three times a year for over 20 years (NIOH,

2002; Saiyed, 2003).

At the request of the Indian Council of Medical Research (ICMR), a three-member team from

NIOH visited the area in August 2001 and recommended following up their visit with an

epidemiological study to investigate the prevalence of disease in school children from the

targeted population and a nearby control population. The field study was conducted from

September 24 to October 7, 2001 with the following objectives (NIOH Report –page 5):

To confirm the reported disease pattern in the exposed populations and evaluate the

magnitude of the problem by comparison with control populations through a well

designed epidemiological study.

To search for etiological factors if the exposed populations show abnormal disease

patterns and generate a hypothesis.

To confirm the presence of endosulfan residues in environmental and biological samples

and estimate their levels.

An initial draft (the First Report) was promised by December 2001 and a final version of the

report (the NIOH Report) was published in July 2002. The final NIOH Report included additional

analyses of drinking water and soil samples collected in June, 2002, after the initial report.

1.2 Information Sources

In completing our review we relied in part on information provided in:

The NIOH Report (NIOH, 2002);

An initial draft of the NIOH Report (hereafter referred to as the “First Report”)(NIOH,

undated); and

A companion paper published in Environmental Health Perspectives (Saiyed et al.,

2003) along with comments on the paper (Abraham, 2004; Indulkar, 2004) and the

author’s response (Saiyed, 2004) published in the same journal.


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In addition, a limited amount of information was provided in response to the request by Mr. B.

Mallesham under the RTI Act 2005. The RTI response included 100 pages of material

including:

93 pages of Gas Chromatography-Electron Capture Detection (GC-ECD) output

corresponding to 2 soil samples analyzed on May 30, 2002 (Soil001.CHI and

Soil002.CHI); 2 water samples analyzed on December 13, 2001 (Water002.CHI and

Water003.CHI); 1 blank sample analyzed on November 21, 2002 (Blank001.CHI); and 4

standard samples analyzed on October 29, 2001 (VK013.CHI and VK014.CHI), October

31, 2001 (VK017.CHI) and December 6, 2001 (Std001.CHI).

1 laboratory notebook page (unsigned and undated) describing the extraction and clean-

up procedure for analysis of endosulfan in soils.

6 partially masked laboratory notebook pages (unsigned and undated) with limited

standard and sample information.


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2 Comments on Analytical Data Quality

The NIOH Report presents the results of analytical chemistry testing used to measure

endosulfan concentrations from samples of soil, sediment, water and human blood. The report

uses these results to make comparisons between the environmental conditions in the Padre

village area, hypothesized to be impacted by aerial applications of endosulfan, and a reference

area (Meenja Panchayath village) approximately 25 km away where this type of application was

reportedly not used. The report uses the results from blood samples to make comparisons

between children from the study versus reference areas. These analytical data are, thus, the

critical foundation upon which the comparisons attempting to link endosulfan to the observations

in Padre village depend.

In our review we identified a number of issues in the way the analytical chemistry tests were

conducted and interpreted that affected the quality and reliability of the analytical data. These

issues were of a nature and extent that preclude the analytical results being considered valid

according to the standards required by governmental regulatory agencies. The results are also

unreliable to the extent that they do not meet the generally accepted standards for use in

guiding scientific interpretations of environmental or public health conditions. These issues

include:

Selected data have been excluded from the NIOH Report without explanation.

Information obtained from NIOH under the Right to Information Act reveals

inconsistencies between the raw data compared to the summary results presented in the

NIOH Report.

The reporting of endosulfan in water and soil at concentrations well below the minimum

detection limit of 1 to 3 ppb and the large numbers of “peaks”reported in the blank and

standard reference samples suggests that random peaks due to electronic noise were

routinely misinterpreted as endosulfan peaks.

Chromatographic peak identification is based solely on retention time –no mass

spectrometry confirmation was performed on any of the study samples because

endosulfan concentrations were too low to confirm by GC/MS.

No calibration data are presented and there is no explanation as to how concentrations

in the samples were calculated from instrument readings.

Results from QA/QC samples and analyses required for the analytical method are not

provided, a condition which frequently results in regulatory agencies rejecting and not

relying upon analytical data.

2.1 Excluded Sample Results

Table 1 of the NIOH Report (page 14) provides levels of endosulfan in water samples collected

in 2001. Data are presented for six samples (3 well, 1 suranga, 1 stream and 1 pond);

presumably, though not specified to be from the study area. Concentrations ranged from

- -endosulfan) and the

degradation product (endosulfan sulfate). While Table 1 contains data for six samples,


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Annexure 6 of the NIOH Report (page 82) notes that “a total of seven samples were collected

from the exposed area (Village Vaninagar Padre) and three samples from the reference area

(Miyapadavu, Meenja Gram Panchayat).”Thus, it appears that selected data, including

apparently all of the results from the reference area, have been excluded from the NIOH Report

without explanation. Excluding the results from the reference area precludes any comparison

between the reference and study areas and any valid scientific representation whether the

conditions differ between the areas.

Similarly, Table 1-A of the NIOH Report (page 15) provides summary data for endosulfan

residues in soil samples collected in 2001. The table indicates that eight samples were

collected from the study area and 2 samples were collected from the reference area. However,

Annexure 6 (page 82) states that “eight soil samples were collected in polyethylene bags from

the exposed area (Village Vaninagar Padre) and three samples from the control area

(Miyapadavu, Meenja Gram Panchayat).”Once again, selected data are omitted from the NIOH

Report without explanation. The fact that only sample results from the reference area were

omitted (i.e., the study and reference areas were apparently treated differently) impairs the

scientifically interpretations that can be reached.

In addition, the description provided in the NIOH Report suggests that the soil samples were

likely not collected at locations useful for comparisons between groups of people from the study

village compared to the reference area. The soil sampling locations described for the study

area were not in the village itself, where the children reside, but from the cashew plantations

750 meters or more upslope from the village (page 12). The specific location relative to the

village is not described for the reference area, but there was reportedly no endosulfan spraying

in this area anyway. Comparisons between soil results from the plantations, in the one case,

versus the village, in the other case, are not reflective of the potential exposure differences for

school children.

2.2 Raw Data from RTI Release Not Matching Results Presented in Report

Raw data obtained from NIOH under the Right to Information Act, 2005 indicate that the

summary statistics for endosulfan levels in soil (mean and standard deviation) presented in

Table 4 on page 18 of the NIOH Report contain numerous calculation errors leading to

inaccurate conclusions as itemized below:

-endosulfan concentration reported for the study area (0.274 ppb) in Table 4

-endosulfan concentration measured for the reference area. The

-endosulfan concentration reported for the study area should be reported as

0.222 according to the raw data. Note that if the values from the raw data were

presented in the table, the soil sample results from the study area (0.222 ppb) would be

shown to be lower than the results from the reference area (0.274 ppb). Currently, the

report refers to this table to substantiate the statement that “the levels were higher in

study area as compared to reference area (page 16).”The raw data contradict this

conclusion.


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Accordingly, total endosulfan concentrations in the top layer of soil, which is the layer

where the greatest potential exposures would occur, are also reported inaccurately.

According to the raw data provided by NIOH, the mean total endosulfan concentration in

the upper soil layer in the study area was 0.242 ppb compared to a mean concentration

of 0.303 ppb in the reference area. Again, the concentration found in the study area for

total endosulfan is lower, not higher than that found in the reference area, similarly

contradicting the study conclusion quoted above.

For the middle layer of soil, the raw data provided by NIOH show that the mean

-endosulfan was 0.104 ppb in the study area compared to 0.184 in the

reference area. Contrary to the study conclusion, this difference is not statistically

significant (p = 0.15) at the 95% confidence level, and the study area is, again, lower

than the reference area. Table 4 has these values switched and incorrectly reports a

value of 0.089 ppb instead of 0.104 ppb as derived from the raw data.

In total, 16 out of 24 summary statistics reported in Table 4 of the NIOH Report (66% of

the values) differed from summary statistics calculated from the raw data sheets

obtained from NIOH under the Right to Information Act, 2005 (see Table 1).

Additionally, simple editing and oversight review of the report during its production

should have revealed the presence of errors and need for careful evaluation. There is

no need for access to the raw data to catch that obviously erroneous information is

presented in this table. For example, total endosulfan levels are reported to be less than

-endosulfan levels in some cases, which is impossible.

Table 1. Differences Between Summary Statistics Reported in Table 4 of NIOHReport and Summary Statistics Calculated from Raw Data

Soil

Layer

Area Mean ±SD Difference

(Yes/No)

Constituent Report Table 4 Raw Data

Top Study -endosulfan 0.274 ±0.161 0.222 ±0.133 Yes

-endosulfan 0.0018 ±0.004 0.018 ±0.039 Yes

Endosulfan sulfate 0.025 ±0.03 0.002 ±0.004 Yes

Total endosulfan 0.030 ±0.18 0.242 ±0.161 Yes

Reference -endosulfan 0.153 ±0.067 0.275 ±0.162 Yes

-endosulfan 0.002 ±0.004 0.002 ±0.005 No




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Middle Study -endosulfan 0.183 ±0.076 0.184 ±0.077 No

-endosulfan 0.005 ±0.001 0.001 ±0.001 Yes

Endosulfan sulfate 0.008 ±0.018 0.008 ±0.018 No

Total endosulfan 0.191 ±0.08 0.192 ±0.089 No


-endosulfan ND 0.001 ±0.003 Yes



Lower Study -endosulfan 0.128 ±0.076 0.128 ±0.077 No

-endosulfan ND ND No

Endosulfan sulfate 0.012 ±0.028 0.012 ±0.029 No



-endosulfan ND ND No

Endosulfan sulfate 0.0005 ±0.001 ND Yes


2.3 Detection Limits

- -endosulfan and

endosulfan sulfate are 1, 1, and 3 pg/ml, respectively. A concentration of 1 pg/ml is equivalent

to 0.001 ppb (1 part per trillion [ppt]) and 3 pg/ml is equivalent to 0.003 ppb (3 ppt). There is no

description as to how these detection limits were determined and the specified levels do not

match those stated twice in other locations in the Final and First Report. The levels on page 84

appear to be the erroneous units for several reasons:

1) Elsewhere in Annexure 6 of the Final Report (page 86) the detection limits for analysis of

- -endosulfan and endosulfan sulfate by GC-ECD are reported as 1, 1 and

3 pg/µl, respectively –equivalent to 1, 1, and 3 ppb.

2) T - -endosulfan and endosulfan sulfate

cited in the First Report (page 61) are given as 1, 1, and 3 pg/µl, respectively –also

equivalent to 1, 1, and 3 ppb.

3) Detection limits of 1-3 ppb would be consistent with the method detection limits reported

elsewhere for water samples using EPA Method 508. Achieving detection limits in the

range of 1-3 ppt (1 pg/ml) with the volumes of material (500 ml for water) used in this

study and no chromatographic cleanup step would be extraordinary, and is highly

unlikely.

4) Further, the records showing computations of concentrations from serum samples

obtained under the RTI request show that a reference standard concentration of 200 ppb

was used in these computations. If the GC/ECD method was actually achieving 1-3 ppt


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detection limits, instead of 1-3 ppb, selecting a standard of 200 ppb would have been

unusual.

There is a clear discrepancy of 1000-fold between the detection limits specified on page 84

compared to those listed on page 86. We have considered the possibility that this discrepancy

is just a lack of clarity related to the conversion between the concentration found in the hexane

diluent compared to the detection limit for the original sample of serum or water. We have also

reviewed information provided in response to the RTI request that suggests the values of 1-3

ppt to be correct due to the mathematical conversion to account for concentration of the extracts

from the samples.

However, based on the detection limits being twice specified by the authors to be 1-3 ppb, the

reasonable expectations of the GC/ECD method, and the chromatograms and information

provided in the RTI response, we have concluded that it is more likely that the statement

indicating 1-3 ppt on page 84 is in error and that 1-3 ppb (1-3 pg/µl) endosulfan in serum or

water is more likely the correct detection limit. If sample detection limits of 1-3 ppt were actually

achieved with the water samples, this would need to be clearly explained and specified.

Throughout the descriptions of the method and detection limits, there is no information at all

regarding extraction methods and detection limits for the solid materials sampled (soil,

sediment, leaves). Detection limits for these types of materials are frequently less sensitive

than those for water samples, however no in-depth comparison can be made and the validity of

the soil and sediment results cannot be assessed without a description of the method, amount

of material extracted and the detection limits achieved.

2.4 Chromatographic Peak Identification

Annexure 6 to the NIOH Report includes a description of “Confirmation Tests”sing gas

chromatography –mass spectrometry (GC/MS). Whereas gas chromatography using an

electron capture detector (GC-ECD –the primary method used to analyze study and reference

population samples) relies on retention time for non-specific compound identification, GC/MS

provides specific identification of compounds through mass spectral analysis.

To attempt to verify the GC-ECD analysis of serum samples from the study population and

document that they were reporting results for endosulfan specifically, the authors analyzed

standard endosulfan samples and serum samples from an individual poisoned with endosulfan

along with serum samples from the study population by GC/MS. However, this approach was

flawed because the levels of endosulfan in serum samples from the study population were

below the limit of detection for the GC/MS instrument, which was reported to be 100 pg/µl or

100 ppb (NIOH Report, page 86). Using this instrument/method, no endosulfan was detectable

in the blood samples from the study population via the specific, GC/MS method.

Moreover, the chromatography column used in the GC/MS analysis (a 30m x 0.25 mm id DB-5

column) differed from the column used in the GC-ECD analyses (a 60m x 0.25 mm HP5

column) such that endosulfan retention times in the GC/MS analysis (ranging from 28.8 to 32.5

minutes) were much shorter than the endosulfan retention times reported for the longer 60 m


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column used in the GC-ECD analyses (ranging from 38.9 to 67.0 minutes). Because the

retention times did not match between the two methods, the GC/MS results could not even

serve to substantiate that the peaks measured in the study population samples via GC/ECD

were likely to be endosulfan. The confirmation method capable of specifically identifying

endosulfan failed to detect this compound in the serum samples from the study population and

the endosulfan standard peaks did not match the retention time peaks quantified as

“endosulfan”from the GC/ECD analyses of the study population.

Note that EPA Method 508 warns in particular about potential interference from phthalate esters

presenting a major problem when using an electron capture detector. Phthalates are

plasticizers (the serum samples were stored in plastic) and are also extremely common at low,

background levels in human blood samples. Because no independent confirmation of peak

identification was performed, there is no way to determine if phthalate ester interference was

contributing to the GC/ECD signal interpreted to be endosulfan.

2.5 Random Noise Fluctuations

The number of distinct “peaks”reported in the chromatographic output provided in the RTI

response (including chromatographic data for the standard samples) ranged from 309 to 486.

Even the blank sample, which is expected to serve as the “zero”sample for the method,

contained integrated areas for 429 “peaks”. Because it is unlikely that the blank contained 429

contaminating compounds, this situation suggests that the software that serves to identify

“peaks”and measure their area was not calibrated to accurately distinguish real peaks from

random electronic noise fluctuation. The frequent reporting of results purported to be

endosulfan in water and soil at concentrations well below the minimum detection limit of 1 to 3

ppb (e.g., see data reported in Tables 1, 2, 3 and 4 of the NIOH Report) suggests that random

electronic noise may have been routinely misinterpreted as endosulfan peaks. In the absence

of independent confirmation of peak identification (performed via either GC/MS or dual column

chromatography), there is no way that apparent peaks corresponding to the thousandth of a ppb

range reported by the authors can be differentiated as actual responses to a specific chemical

versus simply electronic background fluctuation from the detector.

For example, endosulfan concentrations measured in water samples collected in 2001 are

reported in Table 1 (NIOH Report, page 14). Concentrations ranged from 0.0022 to 0.0667 ppb.

These concentrations are well below the reported detection limits of 1 to 3 ppb (currently

interpreted to be the correct final sample detection limits –see Section 2.3, above) and suggest

that the authors may in fact be quantifying random noise fluctuations rather than true endosulfan

-endosulfan levels were greater than -endosulfan levels, which

-endosulfan is the predominant isomer

detected (for example see page 45 of the NIOH Report). This further suggests that the “peaks”

- -endosulfan may have been neither and reflect instrument

electronic noise.

Table 2 of the NIOH Report (page 16) presents levels of endosulfan in drinking water. There

are no units presented in the table, however by comparison to other tables and the First Report,


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it appears likely that the Report is listing values in ppb. Some values are listed as low as 0.0005

and 0.0004. Values of 0.0004 and 0.0005 ppb are below the minimum detection limits reported

in Annexure 6. Similarly, in Table 4 (page 18), which contains a summary of endosulfan levels

in soil samples collected in June 2002, mean endosulfan levels are also reported below the

detection limit.

2.6 Calibration Data

No mention is provided in the report regarding the method used to calibrate the GC/ECD

results, in other words, the calculations needed to convert retention peak areas to

concentrations of endosulfan. Calibration curves are not provided in an Annexure to the report.

Accordingly, there is considerable uncertainty as to how the sample concentrations were

determined.

No calibration curve demonstrating linearity of the detector response is presented.

There is no indication as to how many standards were used to construct the calibration

curve.

The lowest standard on a calibration curve should be approximately ten times the

method detection limit (i.e. around 10 ppb for this study); however, no standard in this

range was apparently run and the lowest standard used was apparently much higher,

i.e., 200 ppb.

While a multi-point calibration curve may have been generated at some point, the 6

partially masked laboratory notebook pages suggest that calculations of sample

concentrations were actually made using single-point calibration with a 1 ppm standard.

To be reliable, among other conditions, single-point calibration should be done using a

standard concentration that is within 20% of the expected sample concentration. In this

case, the study reported calculated concentrations in the sub-ppb range, thousands to

hundreds of thousands of times lower than the single standard (1 ppm) that was used for

calculations. Calculations based on this type of extreme extrapolation are highly

uncertain and such extrapolation would not be acceptable for typical regulatory agency

evaluations of analytical data.

2.7 QA/QC Data

No quality assurance/quality control (QA/QC) data are presented to assess the quality of the

analytical data presented in the NIOH Report. Annexure 6 provides descriptions of the

methodologies used for analysis of endosulfan residues. The method for endosulfan analysis is

said to be based on EPA method Section 5, A, (3), (a) but no actual reference is included and

this terminology appears to reflect some type of typographical error since a method number is

omitted. The method used is actually similar to EPA Method 508 –Determination of Chlorinated

Pesticides in Water by Gas Chromatography with an Electron Capture Detector –which also


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involves liquid/liquid extraction with methylene chloride and analysis by GC-ECD, as done in

this study.1

Results obtained from QA/QC samples are required to evaluate the accuracy and precision of

environmental data collected in a study. Method blanks, field blanks and equipment blanks are

used to evaluate issues with contamination. Laboratory duplicates, field duplicates and matrix

spike duplicates are used to evaluate the precision of the analyses. Surrogate spikes, matrix

spikes and external reference standards are used to establish the accuracy of the results. No

such data are presented in the NIOH Report and there are no indications in the report,

annexures or lab notes provided that such QA/QC testing was completed.

No blank data are presented in the report.

There is no indication that surrogate spikes were used in the analysis.

No matrix spike/matrix spike duplicate results are presented in the report.

There is no indication that any duplicate analyses were performed to assess

measurement reproducibility.

Without presentation of these QA/QC data, there is no way to affirm the reliability of the data

reported in the study. If such QA/QC analyses were not conducted, the study would not meet

the generally accepted scientific standards for environmental chemistry analyses. In this case,

the results would be rejected and not considered usable for scientific interpretations according

to the QA/QC requirements that apply for the U.S. EPA method employed.

1 U.S. EPA. 1995. Method 508 –Determination of Chlorinated Pesticides in Water by Gas Chromatography with an

Electron Capture Detector –Revision 3.1. Edited by J.W. Munch. National Exposure Research Laboratory, Office

of Research and Development, Cincinnati, OH.


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3 Survey Design Comments

The NIOH Report specifies that one of its objectives is to evaluate disease patterns “through a

well designed epidemiological study (page 7).”However, the study described in the report is, in

fact, a simple survey of children in two populations, one from Padre village, near the plantations

sprayed with endosulfan and another from a reference village where such spraying had

reportedly not occurred nearby. The study was implemented rapidly, over approximately 2

weeks in 2001, and involved numbers of participants that ultimately proved too small to provide

useful numbers of the conditions reported. Under generally accepted methods for the design

and conduct of epidemiological studies2

Similar to the analytical chemistry results serving as the foundation upon which comparisons of

environmental conditions rest, the survey and follow-up testing are the foundation upon which

the entire interpretation of potential human health effects relies. And, similarly, this foundation is

inadequate for the uses to which it is stretched.

, surveys can only serve as preliminary tools to generate

hypotheses and do not serve as a basis for reaching conclusions about the causes of health

effects. The study by its own design cannot serve as the basis for conclusion regarding an

etiological link (causation) for endosulfan producing the conditions observed. Even for the

modest goal of hypothesis generation, the design and conduct of the study presented in the

NIOH report was not adequately reliable to produce scientific findings suitable for regulatory or

judicial uses or action.

The validity and strength of epidemiological study results depend on the study design, data

quality and completeness. Factors determining the quality and usefulness of epidemiological

studies include the ability to avoid bias, control for potential confounding variables, and including

sufficient numbers of exposed and non-exposed cases to limit imprecision due to small

numbers. Issues relating to these factors in the NIOH study include:

The study design is a survey, which cannot be used to determine causation, and the

strength of the statistical analyses presented cannot be validated because details on the

population base and how the participants reflect their respective communities are

lacking.

The study fails to account for or discuss the numerous sources of potential bias.

The study fails to address, account for or discuss known and suspected causes of the

numerous outcomes evaluated, known as confounding variables.

Small percentages of surveyed populations participated in some tests conducted for the

study.

2Kleinbaum, Kupper and Morgenstern, 1982; Epidemiological Research: Principals and Quantitative

Methods. Van Nostrand Reinhold; Rothman, Greenland and Lash, 2008; Modern Epidemiology,Lippincott Williams & Wilkins


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3.1 Study Design Considerations

The NIOH Report fails to describe how the actual participants were selected, or to provide the

total number of subjects in each village who would be eligible for inclusion. Without this

information, it is not possible to consider whether those included are representative of children

in the corresponding villages. Thus, it is not possible to ascertain the extent to which selection

forces were operating, resulting in the 619 and 416 in the study and referent groups,

respectively (page 8).

Limited information on the methods of recruitment is provided, and whether these efforts were

comparable and similarly extensive in each location is not discussed. The introduction of the

report describes anecdotal reporting of cases of illness in this area, which would be expected to

sensitize the population in the “study”village as compared to the “reference”village. This

situation can lead to differential reporting and participation in a study, thus biasing findings due

to over-reporting in the “study”village. If different methods were used to recruit participants

between the communities –and if the “study”village was aware of what was under

investigation, any analyses would be subject to potential bias.

Methods to survey/interview participants are described to include staff training (page 9).

However, the staff was apparently not “blinded”to the exposure status of the two groups, and

since the assessments included subjective characterizations, the extent to which observer bias

has been introduced is not known. Given that “abnormalities”were subjectively identified by the

staff, and no criteria are provided that define “major abnormalities,”the potential for intensive

scrutiny or differential inclusion of children in the “study”group is high. This could lead to the

appearance of a greater number of abnormalities in the study group.

This factor is particularly relevant with regard to the Sexual Maturity Rating (SMR) scoring for

male subjects. While Annexure 3 contains tables presumably used to train and guide project

staff on the specific criteria and corresponding scores for girls (page 70-71), no such tables are

provided for boys. The lack of specific and consistent grading categories for boys increases the

likelihood of differential scoring by different examiners and increases the potential of observer

bias. Also, the proforma questionnaire for boys calls for two SMR scores to be recorded, one

for pubic hair and one for “external genitalia and testes (page 65).” No line is provided for a

separate discrete score corresponding to stage of penis development. However, in the

analyses that were presented in the report, separate SMR scores are presented in the following

categories: pubic hair, penis, and testes. Given a direction on the questionnaire form to score

“external genitalia and testes”collectively, there is substantial potential for errors and

uncertainty attempting to separate this into separate scores for penis versus testes development

later in the analysis phase, particularly if the observers were not provided a table with the

scoring criteria.

A questionnaire is provided in the NIOH Report; however, the report states (page 9) that the

parents were interviewed in one of four local languages. The possible misunderstandings and


15

misinterpretations resulting from translation of the health questions into local languages from

English may in fact be substantial, especially if there is no comparable term for the symptoms

listed on pages 60 and 61. Bias can be introduced by the interviewer’s attempts to explain or

translate from English, and if these explanations are more detailed or extensive for the study

group, a bias toward over-reporting can result. However, the report does not indicate how many

participants were issued the questionnaire in each of the languages, nor does the report discuss

any of the challenges involved in the translation or understanding of the symptoms in each of

the languages.

3.2 Confounding Factors and Alternate Interpretations

A significant confounding variable in this study important to a number of the analyses presented

is the inherent age difference between the groups of children from the two villages. The

average age of the reference population is about 10.5 years, compared to 12 years in the study

population (Table 7). In the pre-pubescent and pre-teen years, there can be significant

differences in growth, maturation, development, and learning that are simply a function of how

children develop. Lack of discussion of how an age difference would be expected to affect such

“outcomes”is a serious oversight in qualifying the interpretation of the results presented.

The report also failed to provide data describing the ethnic differences among the populations

studied, which could reflect real differences in socio-economics, nutrition, or genetics that might

affect “health”measures if not considered in the analysis. The limited narrative discussion of

this topic does not provide sufficient information to exclude cultural factors as playing a role in

the differences subsequently found in the survey. The suggestion that because differences in

height and weight were not statistically significant between the groups, the nutritional status is

comparable (page 21) is not substantiated, and cannot be assumed to be a non-factor in other

outcomes assessed. Also, the lack of significance for the reported differences in height and

weight is likely due to the statistical method chosen and failure to stratify the groups by age.

This factor is discussed further below (Section 4.3).

Other possible confounding variables include differential diets, hereditary (genetic) factors

associated with certain ethnicities, quality of teaching between the schools, and family factors

that might differentially influence a child’s performance in school. The lack of acknowledgement

and control for these and other confounding variables is one of the most serious flaws in the

study.

The presentation of “neurobehavioral problems”is similarly lacking a discussion of alternative

explanations for the results reported (Table 9). Again, the children in the two study populations

are of different ages, and thus, the comparison of “learning disabilities”and class retention have

not considered the affect of age, differences in school teaching standards, possible bias on the

part of teachers identifying more “disabilities”among the study village, or family and home

factors that may be affecting learning and classroom behavior. The lack of consideration of age

and other extenuating factors –and possible teacher bias –are also ignored in reporting

findings in Tables 10 and 11, the latter based on very small numbers


16

3.3 Size of Study Groups

Most of the simple tabulations of findings presented in the report show that less than the full

number of participants in each group responded or participated. While incomplete participation

and failure to complete multiple components of a study occur frequently, this limits the

interpretation of what is presented. Also, when findings are only available for small percentages

of study participants, the uncertainty and potential bias become much greater. The report does

not report or attempt to evaluate the scale of uncertainty that resulted from these factors.

Uncertainty analyses are a common component of epidemiological studies and methods to

evaluate the statistical limitations resulting from the low response rates for various endpoints

were readily available. Examples include:

Chromosomal analyses –presented for 2% of reference group and 5% of study group.

Serum samples –obtained from 26% study group and 20% reference group (page 10).

Prevalence of seizure disorders –presented for 41% reference girls and 42% study

group girls (Table 12).

There is additional potential for selection bias as the children for whom blood samples were

taken (page 10) or sexual maturity examinations done are a small sub-set of each study group

(Tables 16 and 18). Any interpretation of comparisons of these small subgroups is not

meaningful, and cannot be assumed to represent the whole eligible study population.

Another issue creating uncertainty related to the selected sub-groups for certain tests is the

difference in participation rates between the study and reference populations. When differing

proportions of groups choose to participate, there is the potential that it is occurring because of

a desire to self-select, either for or against participation. Large differences in participation rate

include:

IQ surrogate testing –57% reference group participation compared to 82% participation

from the Padre village study population (Table 10).

Sexual maturity rating examinations–70% participation from boys in the reference

village compared to 53% participation from boys in the study group (Table 18).

The numbers of children who are compared in the various tables are not sufficient to draw

causal conclusions. The analyses are simplistic and do not all represent the full number of

participants in each evaluation. For example, Table 14 includes less than half of both the study

and referent populations; there is no basis to conclude the majority of girls for whom there is no

information are in fact distributed in the same way as what is given.

The results that are presented stratified by age include findings in some age groups that are too

small to be meaningful. Table 14, for example, presents results for the age distribution of

menstruating girls showing higher numbers and proportions of girls menstruating among the

older girls from Padre village. However, only eight 15-year old girls were included from the


17

reference village, compared with 23 girls of this age from Padre village. Thus, each individual

girl from the reference village has a larger influence on the statistics compared to the Padre

village group. With this type of sample size difference, the status of just a couple of girls in the

reference group has large effect on the apparent difference between the villages. Also, the

statistic presented, an odds ratio, is dependent upon the groups being compared not having

such confounders present. Since the odds ratio compares girls from the reference village that

are younger on average than the Padre village girls, the effect of age can influence comparisons

that have been purported to be affected by environmental factors.

In another example, results of chromosomal analyses presented in Table 22 include 8 reference

and 29 or 21 study participants (for two different measurements). Thus, comparisons cannot be

reasonably characterized as being reflective of the two overall populations –results are

provided from less than 2% of the reference population versus 5% of the study population. With

such a small number from the reference population, the lack of chromosomal abnormalities

among these few children is not a useful basis for comparison. Also, the report states “It may

be noted that the chromosomal abnormalities like dicentric chromosome and chromosome

exchange … were observed in two each of the study subjects.” Drawing note to this

observation implies such small numbers are significant to interpretations when such minimal

observations are not generally accepted as scientifically relevant.


18

4 Comments on Endosulfan Exposure and Effects

One of the specified objectives of the NIOH study was to evaluate etiological factors that could

explain the health conditions observed in the study population; in other words, to evaluate

potential causes. There are specific generally accepted requirements for evaluating potential

causation in toxicological or epidemiological studies and these include the requirement that

purported effects be biologically plausible. In the context of a survey, this means that observed

conditions being evaluated must be consistent with the biological responses that occur from a

putative cause, in this case endosulfan. Also, evaluating causation specifically requires

consideration of possible alternate explanations for observations.

We have evaluated how the conditions and health effects reported in the NIOH Report relate to

the toxicological characteristics of endosulfan and whether the findings reported can

substantiate an etiological (causal) role for endosulfan. We have identified issues with the

relevance of the survey and follow-up testing of a nature and degree that the report does not

meet the standards typical for studies establishing causation for use in regulatory or judicial

actions. The issues include the following areas:

The endosulfan levels measured in blood samples from the study population in Padre

village were within the expected range reported in other studies, making this a weak

candidate cause for conditions in the village.

Age differences between the study group and reference group are an obvious potential

cause of differences in hormone levels that was not fully addressed.

Observations purportedly associated with neurobehavioral conditions and congenital

conditions are insensitive and not toxicologically relevant.

4.1 Endosulfan Levels Consistent With Background

Endosulfan levels in blood samples from the study group are typical of general background

levels found in other populations exposed through routine environmental and dietary sources

(i.e., no specific source such as aerial spraying). In and of itself, this circumstance makes

endosulfan a difficult candidate to establish as the cause of conditions in Padre village, since

there is no indication that exposures are substantially different than experienced elsewhere.

Additionally, the computation of values from the reference population generated levels that are

unusually low in comparison to expected background levels. Together, these circumstances

suggest it is more likely there is something out of the ordinary about the computed values from

the reference village than the results from Padre village. Comparisons made between these

reference group results and the results from Padre village are, thus, weakened by the

uncertainty inherent in the former being inconsistent with other scientific studies.

Endosulfan testing results were presented for blood serum samples collected from sub-groups

of the children studied in each village. These sub-groups were not selected randomly and are

stated to be an outcome of the willingness of parents and children to consent to blood sampling.

Results are reported for approximately 1 out of 5 children studied in the reference village and 1


19

out of 4 children studied in Padre village. The difference in participation rates suggests that

parents in Padre village were more likely to consent, potentially because they were more

sensitized to concerns regarding endosulfan. This highlights the potential for selection bias

affecting the outcome of comparisons based on the serum sample results.

The NIOH Report references Table 5 stating that “endosulfan residues were found in 85% and

78% of female and male subjects, respectively of study area; whereas they were found in 34%

and 29% of female and male subjects in the reference group (page 16).” First, there appears to

be a typographical error in Table 5, because the percentages stated to be from the study area

are actually shown in the table as being from the reference area and vice versa. In this case,

based on comparison of the subgroup sizes subsequently reported in the published version of

the study (Saiyed et al., 2003), it appears that the error is in the table heading, as opposed to

the text explanation. Additionally, this statement fails to make clear that the percentages do not

actually apply to the overall subject groups, but only the non-randomly selected subgroups.

Thus, endosulfan was not actually detected in 85% of the 619 subjects from Padre village,

rather in 85% of the 97 children who provided blood samples.

The NIOH Report presents serum levels of -endosulfan, -endosulfan and endosulfan sulfate

grouped by gender of the children (Table 6). No units are shown or mentioned in the text,

however, by comparison to the published version (Saiyed et al., 2003) it appears that they are

likely presented in parts per billion. Combining the males and females, the average result for

the group from the reference village is approximately 1 ppb, while the overall average for the

study group from Padre village is between 9-10 ppb.

Interestingly, it is the results from the reference group that appear unusual compared to other

studies. Because of its wide usage in parts of the world, there are routinely found background

levels of endosulfan in human blood, even in the absence of specific exposures such as the

aerial spraying being investigated around Padre village. Results from a study conducted in four

Punjab villages found average levels of -endosulfan and -endosulfan of approximately 5 ppb

in human blood samples (Mathur et al., 2005). A study in Spain reported an average total of

approximately 9 ppb for -endosulfan, -endosulfan and endosulfan sulfate combined in

umbilical cord blood from newborns, and even higher levels of other endosulfan metabolites not

accounted for in the NIOH study (Cerrillo et al., 2005). These results suggest that background

levels should have been expected to be in the 5-10 ppb range where exposures were general

environmental and dietary sources of endosulfan. The results from the Padre village group

appear to be within the range of routine background endosulfan levels found in other

populations. The reported values for this village do not stand out as being obviously elevated

due to exposures from the aerial spraying patterns on the plantations in the area.

Conversely, the reported average values of approximately 1ppb in the reference area appear

low relative to expected background and suggest the need to obtain the raw data from these

measurements to ascertain how non-detect values were handled in computing the averages.

The report does not specify whether averages were computed only using detected values. With


20

more than 2/3rd non-detected samples from the reference village, the approach used to deal

with censored values is important to consider.

Further information suggesting the potential impacts of including samples below detection limits

in the analyses emerges by referring to the First Report. Table 2 (page 12) in the First Report

provides a listing of individual serum sample results for 22 individuals from Padre village. This

version of the report specifies that at that point, 170 children from Padre village had been

sampled, but no individual nor summary information is presented for the remaining children. For

serum samples, the specified detection limit was 3 ppb for endosulfan sulfate. Three children

are listed to have values lower than this (1.57, 2.79 and 2,9 ppb) in the table. The quantification

of these results is uncertain if they are below the minimum detection limit for the method. In the

Final Report, no individual measurements are provided, just summary information such as the

mean, which is influenced by the manner in which censored values (below detection limits) are

included. The handling of the reported values below detection limits is not specified. They

should have been treated as “non-detects.”

4.2 Samples Excluded from Journal Publication

The NIOH study is dated 24 July 2002. A journal article presenting some of the data and results

from the study, particularly focusing on the results for the males, was submitted for publication

on 10 February 2003 (Saiyed et al., 2003). With regard to the blood sample results, there are

notable differences in the datasets presented in these two documents. While endosulfan blood

levels for 97 boys from Padre village are summarized in the NIOH report, the published article

includes results from only 70 boys from the study village. The average total endosulfan level in

the published version is 7.47 ppb, compared to 8.71 ppb in Table 6 of the NIOH Report. This

suggests that the endosulfan results from the 27 boys excluded from the journal publication

were actually a bit higher, on average. The omission of results from these boys is curious since

it reduces the apparent elevation of endosulfan in boys from Padre village. This suggests that

there was some compelling reason that the results from these 27 boys were not considered

suitable for publication, opening up the possibility that data quality issues were recognized

between the release of the NIOH Report and subsequent journal publication. In comparison,

results from 48 boys from the reference village appeared in the NIOH Report and only 3 of

these were excluded in the dataset for the publication, which included results from 45 reference

village boys. The outcome that the exclusions occurred to such differing degrees between the

groups further suggests there was some factor specific to the detected levels reported in the

NIOH Report for the study village that was reconsidered. Also, the published article does not

disclose that the results presented are a selected subset of samples from the larger NIOH study

or explain the basis for excluding samples.

Another unusual quantitative outcome is the reporting of the frequency of detecting endosulfan

among the tested samples stated in the journal article, “endosulfan was detected in serum

samples of 78% of the children in the study group and 29% of the children in the control group

(page 1961).”These are exactly the same values stated in the NIOH Report and presented in

Table 5, albeit apparently reversed. It seems extraordinarily unlikely that the percentages of

samples with detected levels of endosulfan could have been identical once 27 study group and


21

3 control group boys were dropped. This raises a question as to whether the published

statement regarding the frequency of detection is accurate based on the smaller dataset or

whether it might have referred to the larger dataset from the NIOH study, which was not

disclosed in the article. Presenting summary statistics, such as the frequency of detection, using

a different number and group of samples than those described in the materials and methods

section of a submitted journal article would be inconsistent with expected transparency in

scientific publishing.

4.3 Obvious Alternate Explanations Not Discussed

The data presented in Table 7 show that the girls from Padre Village were older, taller and

heavier than the corresponding groups from the reference village. The average age of the study

group (12.0 years) is a full year-and-one-half older than the average age of the reference village

girls (10.5 years). Particularly at these peri-pubescent ages, such a difference is an obvious

alternative factor in hormone levels. Additionally, the girls from Padre village were 19% heavier

on average than the girls from the reference village (30.8 kg vs. 25.9 kg). This difference also

suggests the obvious possibility that the overall group of girls from Padre village was at a more

advanced stage of puberty.

The NIOH report does acknowledge the age difference stating, “the mean age of the study

group is higher as compared to reference population (page 19).” However, the report also goes

on to conclude that “the sex-wise distribution is comparable in study and reference groups

(page 19).” Such a conclusion in light of a 1.5-year age difference and 5 kg weight difference

does not appear adequately supported.

The NIOH study reports that levels of Luteinizing Hormone (LH), progesterone and estradiol

were higher in the female study group from Padre village compared to the reference village

(Tables 26, 29 and 30). This is the outcome that you would expect with such hormones from an

older group of peri-pubescent girls and it substantiates that the position stated in the report that

the groups from the different villages can be considered comparable is subject to challenge.

Both the NIOH Report and the subsequent journal publication (Saiyed et al., 2003) focus

extensively on results relating to male reproductive development, particularly testosterone levels

and SMR scoring for the boys. However, results relating to both of these parameters are also

not adequately considered with regard to alternate explanations. For the SMR scoring, the

failure to provide scoring criteria, failure to “blind”the observers, and small differences in a

subjective score indicate the obvious potential for differences to be explained by observer bias.

For the testosterone results, the report and publication acknowledge that age is a controlling

factor, but suggest that residence in Padre village is also a statistically relevant factor.

However, the statistical testing and information that would allow a reviewer to evaluate the

relative importance of these two different causes is not presented.

SMR scoring involves assigning a categorical value of 1 to 5 based on certain observable

characteristics related to sexual maturity. In the report, the differences reported fall

predominantly between scores of 2 and 3 and separate scores for pubic hair, penis and testes


22

are presented in tables and figures. The values for penis development cannot be considered

reliable because the questionnaire provided to examiners did not even call for a separate score

for penis development. It is not clear how the analysts obtained such values.

The scores and differences between the study and reference groups for testes development

illustrate the subjective nature and high uncertainty of using this parameter. While the criteria

employed by project staff for boys were not disclosed in the report or its annexures, a common

version of the Tanner SMR criteria relating to development of the testes and scrotum is:

SMR Score 2 –Enlargement of scrotum and testis, reddening and change of texture of

scrotum

SMR Score 3 –Growth of testes and scrotum

Particularly for examiners seeing a patient for the first time, differentiating between whether the

boy qualifies for a score of 3 versus 2 is obviously subjective and uncertain.

The differences tabulated (Table 18) and plotted (Figure 3) between the study and reference

groups show that the Padre village boys aged 12, 13, 14, and 15 scored approximately 0.5 SMR

point lower than the corresponding aged boys from the reference village. In other words, the

difference reported to be significant corresponds to something about halfway between

“enlargement”versus “growth”of the testes and scrotum. Also, there are small numbers of boys

of each age, ranging from only 5 to 14 with SMR scores of 2 or higher. In the context of these

minor differences, unintended observer bias is an obvious possibility that should have been

addressed. Providing consistent scoring charts and using examiners blinded to the village of

residence for each boy were critical when interpretations depended on such fine distinctions.

The NIOH Report states “levels of testosterone were lower in the study group as compared to

reference population in the same age group (page 37).” This statement is not a precise

representation of the results in Table 28 because for boys of some ages, the levels were

actually lower in the reference group. Looking at boys older than 10, the table shows that the

11 year olds were essentially equivalent between the villages, the reference village boys had a

clearly lower average testosterone level for ages 12 and 15 and the Padre village boys had a

clearly lower average for ages 13, 14 and 16. And, the 16-year old group from Padre village

included only two boys. This type of inconsistent pattern based on small numbers is not a clear

indication of effects in Padre village. Statistical analyses intended to help differentiate between

the obvious effect of age on testosterone production and a possible effect of location (i.e.,

village of residence) were apparently conducted. However, the summary information presented

is not sufficient to determine the relative contribution of these two factors or to reanalyze the

data to confirm the report findings.

4.4 Recorded Observations Not Biologically Relevant

The conclusions presented in the NIOH report specify that the study identified a higher

prevalence of neurobehavioral disorders, male reproductive system abnormalities, and

congenital malformations in females in the study group from Padre village. Interpreting the


23

findings of the study in these terms is inconsistent with both the results obtained and the

generally accepted meaning of these terms.

The results apparently categorized to indicate neurobehavioral disorders were 1) differential

scholastic performance, 2) differential performance in a test to draw a man, and 3) abnormal

behavior reported by teachers. None of these parameters are directly or uniquely indicative of a

biologically based neurobehavioral disorder. The report states “the prevalence of arrogant and

aggressive behavior and restlessness were higher in the study group as compared to the

reference population (page 22).” According to reporting by teachers for the 619 Padre village

children, 1.8% of them were arrogant, 1.3% were aggressive and 0.3% were restless. The

comparison made is to reporting from the teachers of the 416 reference village children among

whom there was not a single restless or aggressive child noted and only one child reported to

be arrogant. Such a finding is clearly a difference without meaning in the context of declaring

children to have “neurobehavioral disorders.”Given the awareness of the Padre village teachers

of the concerns regarding endosulfan, reporting bias is an obvious consideration, along with

some type of motivation to underreport that may be affecting the teachers of the apparently 415

near perfect children from the reference village. These endpoints are not appropriately sensitive

to identify actual clinically relevant disorders and are not indicators of endosulfan toxicology.

In addition to the reported differences in testosterone levels and SMR scoring discussed above,

the other observations apparently interpreted as male reproductive system abnormalities in the

report include two conditions reported in Table 12 –undescended testes and congenital

hydrocele. Two cases of undescended testes were reported among 361 boys from Padre

village, amounting to 0.55% of the boys. This is a relatively common condition with a

prevalence of around 1% in boys over age 2. Higher rates are seen among newborns, but they

commonly resolve prior to age 2. The number of cases seen in Padre village is, thus, not

higher than would be expected. Curiously, Table 12 shows the fraction 2/361 and

parenthetically 1.55%. This incorrect calculation overstates the percentage by almost threefold

and makes the table entry appear higher than the general population rate of around 1%.

Congenital hydrocele is another common condition at birth, occurring in approximately 1-2% of

boys. Hydrocele refers to fluid accumulation around the testes and congential cases also

frequently resolve as the testes complete their descent and the scrotum becomes isolated from

the abdominal cavity, allowing the fluid to resorb. The report lists four cases of congenital

hydrocele, meaning the condition was present at birth, among 361 Padre village boys. This

amounts to 1.1% of the group, again not higher than expected.

In the published journal article version of the study (Saiyed et al., 2003), cases of a third

condition, congenital inguinal hernia, are included along with undescended testes and

congenital hydrocele, even though there is one case in each of the study and reference

populations. The article reports a total of 6 cases among these three conditions (omitting one of

the hydrocele cases for some reason) and, then computes a prevalence of 5.1% in Padre

village (abstract, page 1958) by revising the group size to report that they were observed among

117 subjects (6/117). Changing the denominator in this manner between the report version and


24

published version, and adding an extra case to the numerator from another condition for which

there is clearly no difference among the study and reference populations, thus computing a

higher prevalence rate from the same underlying study, is inconsistent with expectations of

scientific publishing.

While the conclusions of the report specify differences in female congenital conditions

collectively to be important, review of Table 12 shows that the only significant difference

between the two villages was noted for the non-specific grouping “congenital heart disease.”

Nine cases were noted among 258 girls from Padre village. Only one case was noted among

183 girls from the reference village. By segregating the girls in this manner the report shows an

apparent difference. However, congenital heart diseases in general are not particularly linked to

gender. And, among the boys, the prevalence of congenital heart disease is 3 times higher in

the reference village than in Padre village according to Table 12. The observations among the

boys are, thus, contradictory to the pattern observed with the girls. Since there is no biologically

reasonable basis or research suggesting that endosulfan affects cardiac development in

opposing manners between males and females, the observations collectively grouped as female

congenital conditions are not, in fact, relevant to establishing endosulfan as the cause. This is a

case where the difference is more likely a matter of chance related to the small number of

reported cases.


25

5 Conclusions

The NIOH report draws a series of conclusions including that there is a significantly higher

prevalence of neurobehavioral disorders, congenital malformations in females, and male

reproductive abnormalities in the Padre village area. The report also concludes that sufficient

information has been considered such that endosulfan exposure from aerial spraying in the

cashew plantations upslope from the village is the “most probable”cause of the health

conditions documented in the report.

These conclusions are not supported by the study design, methodologies, or results of the

investigation that was completed. The conclusions outreach the stated objectives of the study,

which specify that the survey approach was intended to serve to generate a hypothesis

regarding potential etiological factors. This objective was theoretically attainable, however the

survey method employed cannot, as a matter of generally accepted epidemiological science,

serve as the basis for determining causation from a single environmental factor. The design

and methods are suited solely for preliminary hypothesis generation. Therefore, the survey

could not, in the first place, support the conclusion reached regarding endosulfan as the

probable cause.

Review of the Final Report, the First Report, the information provided subsequent to an RTI

request and available scientific literature lead us to conclude that this investigation is impacted

by analytical limitations; computational errors; reporting and documentation errors and

omissions; and inadequately substantiated interpretations to such an extent that it is not

scientifically reliable. The uncertainties are sufficient that results from this investigation would

frequently be rejected by environmental regulatory agencies and excluded from consideration of

regulatory or judicial actions.

We recognized serious limitations in the analyses for endosulfan conducted on environmental

and blood samples. The failures to provide QA/QC documentation or confirm the presence of

endosulfan with a chemical-specific method and the implications of the large number of

apparent chromatographic “peaks”in the blank create uncertainties with regard to whether

endosulfan was even the compound detected in some samples with low reported

concentrations. The presentation of summary data alone, without appendices containing the full

set of results, makes it impossible to recreate the computed means and variances and ascertain

how non-detected values were included.

The survey approach used to document health conditions is subject to substantial sources of

potential bias and did not include steps to either eliminate or specifically recognize the impact of

biases. Tests for confounding factors were not included and the simple summary statistics

compared are not sufficient to determine the potential interactions among various factors

affecting health. The conditions were grouped and categorized such that the seriousness and

prevalence of conditions was overplayed. Endpoints that are highly subjective, such as

arrogance or aggressiveness reported by a teacher, were represented to be neurobehavioral

disorders. Quantitative measurements such as hormone levels were compared using summary

statistics that did not account for the obvious and expected effect of age in pubescent subjects.


26

Congenital conditions reported as male reproductive effects were actually found among the

boys from Padre village at rates below the prevalence expected in the general population.

The reporting of the investigation also failed to meet standards of transparency and clarity

expected in scientific reports. Sample results were excluded and the number of subjects

participating was manipulated between different versions of reporting the same study.

Information presented in a followup publication from the study was selected in ways that inflated

the apparent prevalence of congenital conditions in Padre village. Summary statistics were

computed from varying numbers of subjects without explanation and there were serious

discrepancies in some cases between the raw data obtained through a Right to Information

request and the numbers presented in the report and publication.

In conclusion, the NIOH Report was necessarily limited, as any investigation, by the practical

issues of timing, participation and study design. In addition, however, the uncertainties of the

investigation were not made clear and its lack of suitability, as a brief, preliminary hypothesis-

generating study, for informing regulatory or judicial actions was not acknowledged. In contrast,

the conclusions presented, in particular the finding that endosulfan application is the most

probable causative factor for conditions reported, amount to stretched and selective

interpretations of the results obtained. The study is not a valid means to support such a

conclusion. The report is not an appropriate or complete scientific representation of the study

conduct and findings. Substantive corrections, expanded disclosures and further analyses are

necessary before the report would meet the expected standards for scientific reporting.


27

References

Abraham, C.C. 2004. Endosulfan’s Effects: Omissions and Flawed Data. EnvironmentalHealth Perspectives 112(10): A538.

Arrebola, F.J., J.L. Martinez Vidal and A. Fernandez-Gutierrez. 2001. Analysis of endosulfanand its metabolites in human serum using gas chromatography-tandem mass spectrometry.Journal of Chromatographic Science. 39(5): 177-182.

Cerrillo, I., Granada, A., Lopez-Espinosa, M-J., Olmos, B., Jimenez, M., Cano, A., Olea, N., andM.F. Olea-Serrano. 2005. Endosulfan and its metabolites in fertile women, placenta, cordblood, and human milk. Environmental Research 98:233-239.

Goyal, R.K. and H.G. Koshia. 2011. Report of the Committee to Evaluate the Safety Aspects ofEndosulfan. Report submitted to Department of Health & Family Welfare, Government ofGujarat, Gandhinagar, Gujarat, India. March 15.

Gupta, P.K. and R.C. Gupta. 1979. Pharmacology, toxicology and degradation of endosulfan.A review. Toxicology 13: 115-130.

Indulkar, A.S. 2004. Endosulfan’s Effects: Inaccurate Data. Environmental HealthPerspectives 112(10): A538-A539.

Mathur, H.B., Agarwal, H.C., Johnson, S., and N. Saikia. 2005. Analysis of Pesticide Residuesin Blood Samples from Villages of Punjab. Report of the Centre for Science andEnvironment, Pollution Monitoring Laboratory. New Dehi. March 2005.

National Institute of Occupational Health (NIOH). 2002. Final Report of The Investigation ofUnusual Illnesses Allegedly Produced by Endosulfan Exposure in Padre Village of KasargodDistrict (N. Kerala). Report submitted to the Honorable National Human Rights Commissionby Indian Council of Medical Research. Ahmedabad-380016. July 22.

National Institute of Occupational Health (NIOH). Undated. Report of The Investigation ofUnusual Illnesses Allegedly Produced by Endosulfan Exposure in Padre Village of KasargodDistrict (N. Kerala) (First Report). Report prepared by Indian Council of Medical Research.Ahmedabad-380016.

National Institute of Occupational Health (NIOH). 2010. September 24 Letter Report from P.C.Yadav (NIOH) to B. Mallesham re: Information Provided under the RTI Act, 2005.

Saiyed, H., A. Dewan, V. Bhatnager, U. Shenoy, R. Shenoy, H. Rajmohan, K. Patel, R.Kashyap, P. Kulkarni, B. Rajan and B. Lakkad. 2003. Effect of endosulfan on malereproductive development. Environmental Health Perspectives 111(16): 1958-1962.

Saiyed, H.N. 2004. Endosulfan’s Effects: Saiyed’s Response. Environmental HealthPerspectives 112(10): A539-A541.

review of nioh study of endosulfan in kerala, india

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