review of nioh study of endosulfan in kerala, india
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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 USArdemott@environcorp.com
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
Scientific Review –NIOH Endosulfan Study
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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.
Scientific Review –NIOH Endosulfan Study
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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
Endosulfan sulfate 0.007 ±0.012 0.026 ±0.039 Yes
Total endosulfan 0.162 ±0.08 0.303 ±0.187 Yes
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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
Reference -endosulfan 0.089 ±0.096 0.104 ±0.098 Yes
-endosulfan ND 0.001 ±0.003 Yes
Endosulfan sulfate 0.007 ±0.012 0.004 ±0.007 Yes
Total endosulfan 0.096 ±0.091 0.109 ±0.098 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
Total endosulfan 0.106 ±0.085 0.141 ±0.087 Yes
Reference -endosulfan 0.0623 ±0.06 0.086 ±0.053 Yes
-endosulfan ND ND No
Endosulfan sulfate 0.0005 ±0.001 ND Yes
Total endosulfan 0.062 ±0.059 0.086 ±0.053 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
Scientific Review –NIOH Endosulfan Study
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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
Scientific Review –NIOH Endosulfan Study
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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.
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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.
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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.
Scientific Review –NIOH Endosulfan Study
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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.
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