National Health and Nutrition Examination Survey

Atrazine is a widely used chlorotriazine herbicide, active against broadleaf and grassy weeds, and applied both pre-and post-emergent to agricultural land for crops such as corn and sorghum. It is also used as a non-selective herbicide. In areas where atrazine is used, it may be detected pesticides in surface and ground waters. Atrazine does not bioaccumulate, but it degrades slowly in soils to dealkylated products, which may last for several months. Atrazine use has been progressively restricted because of concerns that drinking water sources may become contaminated. Human exposure may be evaluated by measuring urinary metabolites, but the dealkylated metabolites can result from metabolism of other chlorotriazine herbicides, including simazine, propazine, cyanazine. The dealkylated atrazine metabolites and hydroxyatrazine can also occur in the environment from breakdown of atrazine. Therefore, detection of the dealkylated metabolites in urine may reflect exposure to atrazine, another chlorotriazine herbicide, environmental breakdown products of one or more chlorotriazine herbicides, or any combination of these. Because atrazine is widely used in the U.S., measuring urinary atrazine and its metabolites is important to evaluate human exposure and potential for health effects.

Examined participants aged 6 years and older from a one-third sample.

Urine specimens are processed, stored, and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, and Centers for Disease Control and Prevention for analysis. Vials are stored under appropriate frozen (–30°C) conditions until they are shipped to National Center for Environmental Health for testing.

 Atrazine (6-chloro N-ethyl-N’-(1-methylethyl)-1,3,5-triazine-2,4-diamine; CAS Number: 1912-24-9; AAZ) is a triazine herbicide used to kill weeds, primarily on crops such as sugarcane, corn, pineapples, sorghum, and macadamia nuts. It has also been used on evergreen tree farms, evergreen forest re-growth, highway and railroad rights-of-way, and turf application. Atrazine was the most used active ingredient in agriculture in the United States for nearly two decades, until it was replaced by glyphosate . Regardless, the amount of atrazine applied annually has remained relatively constant with tens of million pounds applied annually in the United States.

AAZ enters into the environment, particularly soil and water. Its primary degradation can occur via soil bacteria and abiotic processes with an environmental half-life of a few weeks to several months (ATSDR 200. AAZ and its degradation products tend to migrate out of the soil into water systems including surface runoff to streams, river, and lakes, or deep ground water systems and aquifers creating a potential for human exposure.

The AAZ metabolism may differ based on the exposure scenario. Occupational exposures may result in increased exposure to atrazine itself while environmental exposures are likely dominated by the dealkylated environmental degradates which are presumed to be biologically active if they retain the chlorine atom. Thus, measurement of multiple potential metabolites is necessary to best assess exposures to atrazine and its related degradates.

The method uses solid phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) for quantifying atrazine and its metabolites and/or hydrolysis products. The method relies on automated off-line SPE to pre-concentrate the target compounds while reducing or eliminating most of the urine matrix potential interferences to increase the overall sensitivity and specificity. Off-line SPE is coupled with on-line two-dimensional (2D) HPLC combining cation exchange (SCX) and reversed phase (RP) chromatography in one integrated HPLC system followed by isotope dilution tandem mass spectrometry for the quantitative determination of the target analytes. Also, for high exposure studies (i.e., animal toxicology) all target compounds can be measured by injecting a small amount (10 µl) of sample directly into the HPLC-MS/MS system without a pre-concentration step.

Detailed instructions on specimen collection and processing can be found in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM).

Refer to NHANES 2007 - 2008 Lab Methods for Atrazine and metabolites laboratory for a detailed description of the laboratory method used.

Desethyldesisopropylatrazine (DCZ), Desisopropylatrazine (SIS), Desethylatrazine (DTZ), Atrazine (AAZ), Desisopropilatrazine mercapturate(SISM), Atrazine mercapturate (ATZ) (September 2015)

The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Act mandates.

Detailed instructions on specimen collection and processing are discussed in the NHANES Laboratory Procedures Manual (LPM).

Read the General Documentation on Laboratory Data file for detailed QA/QC protocols.

Mobile Examination Centers (MECs)

Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured quality assurance evaluation during unscheduled visits to evaluate both the quality of the laboratory work and the quality-control procedures. Each laboratory staff person is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback are given to each staff. Formal retraining sessions are conducted annually to ensure that required skill levels were maintained.

Analytical Laboratories

NHANES uses several methods to monitor the quality of the analyses performed by the contract laboratories. In the MEC, these methods include performing blind split samples collected on “dry run” sessions. In addition, contract laboratories randomly perform repeat testing on 2% of all specimens.

NCHS developed and distributed a quality control protocol for all the contract laboratories which outlined the use of Westgard rules (Westgard et al, 1981) when running NHANES specimens. Progress reports containing any problems encountered during shipping or receipt of specimens, summary statistics for each control pool, QC graphs, instrument calibration, reagents, and any special considerations are submitted to NCHS quarterly. The reports are reviewed for trends or shifts in the data. The laboratories are required to explain any identified areas of concern.

All QC procedures recommended by the manufacturers were followed. Reported results for all assays meet the Division of Environmental Health Laboratory Sciences’ quality control and quality assurance performance criteria for accuracy and precision, similar to the Westgard rules (Caudill et al., 2008).

Read the General Documentation on Laboratory Data file for detailed data processing and editing protocols. The analytical methods are described in the Description of Laboratory Methodology section above.

Refer to the 2007-2008 Laboratory Data Overview for general information on NHANES laboratory data.

Subsample weights:

Atrazine and metabolites were measured in a one third subsample of persons 6 years and older. Special sample weights are required to analyze these data properly. Specific sample weights for this subsample are included in this data file and should be used when analyzing these data.

Please refer to the NHANES Analytic Guidelines and the on-line NHANES Tutorial for further details on the use of sample weights and other analytic issues.

 NHANES Survey Design:

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2007-2008 Demographics File contains demographic data, health indicators, and other related information collected during household interviews as well as the sample design variables. The recommended procedure for variance estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU, respectively) in the demographic data file.

This laboratory data file can be linked to the other NHANES data files using the unique survey participant identifier (i.e., SEQN).

Detection Limits:

The detection limits were constant for all of the analytes in the data set. Two variables are provided for each of these analytes. The variable named URDATZLC indicates whether the result was below the limit of detection. There are two values: “0” and “1.” “0” means that the result was at or above the limit of detection. “1” indicates that the result was below the limit of detection. For analytes with analytic results below the lower limit of detection (i.e., URDATZLC=1), an imputed fill value was placed in the analyte results field. This value is the lower limit of detection divided by square root of 2 (LLOD/sqrt [2]).

The other variable named URX___ provides the analytic result for that analyte.

The lower limit of detection (LLOD in ug/L) for the atrazine and metabolites is: 

Please refer to the NHANES Analytic Guidelines and the on-line NHANES Tutorial for further details on the use of sample weights and other analytic issues.