California Environmental Protection Agency 
Department of Pesticide Regulation 
Environmental Monitoring and Pest Management 
1020 N Street, Room 161 
Sacramento, California 95814-5624 
STUDY 155: PROTOCOL FOR MONITORING ACUTE AND CHRONIC TOXICITY IN THE SAN JOAQUIN RIVER WATERSHED, WINTER 1996-97 
December 9, 1996 

I. INTRODUCTION 

In the San Joaquin Valley, the organophosphorus insecticides diazinon, chlorpyrifos, or methidathion are generally applied with a dormant oil on nut and stone fruit trees to control peach twig borer, San Jose scale, European red mite, and brown mite pests. The best time to achieve control of these pests is December through February, when trees are dormant and better pesticide coverage is possible (Zalom et al., 1995). This dormant orchard spray application period, however, coincides with seasonal rainfall. Thus, these pesticides have the potential to wash off target areas and migrate with runoff waters to the San Joaquin River. This protocol is designed to monitor toxicity of pesticides in the San Joaquin River watershed during winter months when dormant insecticidal sprays are being applied to orchards.

From 1988 to 1990, the Central Valley Regional Water Quality Control Board conducted an aquatic toxicity survey in the San Joaquin Valley. Surface water samples collected from certain reaches of the San Joaquin River watershed during this survey were found to have been acutely toxic to the water flea, Ceriodaphnia dubia (Foe and Connor, 1991). The cause of toxicity was not determined but was attributed to pesticides in general. Further study was conducted in the Valley during the winter of 1991-92, and the resultant toxicity was attributed to the presence of chlorpyrifos and diazinon (Foe and Sheipline, 1993; Foe, 1995; Kuivila and Foe, 1995). The toxicity found in these studies was found to have been in violation of the Central Valley Regional Water Quality Control Board's narrative water quality objective (Foe, 1995). The toxicity objective states that, "All waters shall be maintained free of toxic substances in concentrations that produce detrimental physiological responses in human, plant, animal, or aquatic life" (CVRWQCB, 1994).

Additionally, the Department of Pesticide Regulation monitored the San Joaquin River watershed during the winters of 1991-92 and 1992-93 and reported the detection of chlorpyrifos, diazinon, and methidathion in 10, 72, and 18 percent of the 108 water samples collected, respectively (Ross, 1992 and 1993). Of these positive samples, 2, 13, and 1 percent exceeded the LC50 for C. dubia, respectively, indicating potential acute toxicity. In addition, diazinon detections in the San Joaquin River at Vernalis ranged from 0.148 to 1.07 mg/L, on 12 consecutive days, and the authors concluded that chronic toxicity due to diazinon might be problematic at this site (Kuivila and Foe, 1995). In addition, dormant spray insecticides, at levels acutely toxic to test organisms, have been reported in Orestimba Creek, a tributary to the San Joaquin River, during the 1992-93 dormant spray period (Domagalski, 1995). Consequently, programs to reduce the mass of dormant orchard spray insecticides leaving target areas are currently under investigation by the Department of Pesticide Regulation and growers (Ross, 1995; Ando, 1996; Anonymous, 1996; Biermann, 1996).

In this study we will look at acute toxicity to C. dubia in a small watershed where the discharging waters do not contain municipal or industrial contaminants. We will also investigate the potential for chronic toxicity in a reach of the San Joaquin River downstream from major orchard and tributary inputs in the watershed. Long term monitoring of acute and chronic toxicity will help scientists at the Department of Pesticide Regulation evaluate the effectiveness of programs designed to decrease the runoff of dormant spray insecticides.

II. OBJECTIVE 

The objective of this study is to monitor the occurrence of toxicity, both acute and chronic, and chlorpyrifos, diazinon, and methidathion levels in the San Joaquin River watershed during the dormant spray season. A companion study will be conducted to monitor toxicity and pesticide levels in the Sacramento River.

III. PERSONNEL 

This project will be conducted by the Environmental Hazards Assessment Program (EHAP) under the general direction of Don Weaver, Senior Environmental Research Scientist (Supervisor). Key personnel are listed below:

Project Leader: Kevin Bennett

Field Coordinator: Andrea Hoffman

Senior Scientist: Lisa Ross

Statistician: Terrell Barry

Contractor (toxicity tests): Charlie Huang, California Department of Fish & Game

Chemists: Jorge Hernandez, Jean Hsu, and Hsiao Feng,

California Department of Food & Agriculture

Agency and Public Contact: Pat Dunn

Questions concerning this monitoring project should be directed to Pat Dunn at

(916) 324-4100. Fax: (916) 324-4088.

IV. STUDY PLAN

The San Joaquin River extends approximately 215 km from Friant Dam to Stevinson where flows are intermittent. The flow from Stevinson to Vernalis (about 97 km), is perennial. Acute toxicity sampling will be conducted at Orestimba Creek, a western tributary to the San Joaquin River, as this site receives runoff that is predominantly agricultural (Figure 1). Sampling for chronic toxicity will be conducted on the San Joaquin River at Vernalis, as this site receives discharges from all of the River's major agricultural tributaries, including the Merced, Tuolumne, and Stanislaus Rivers. Discharge records for both the Orestimba Creek and Vernalis sampling sites are available from collocated gauging stations. This information will be used to correlate any changes in chemical concentrations to fluctuations in flow and may be useful for modeling efforts, should they be undertaken.

Monitoring will commence prior to the onset of the dormant spray season (December 1996) and continue through the end of March 1997. Background samples will be collected for one week, beginning prior to dormant spray applications, then monitoring will resume once applications have began in the watershed. Monitoring will proceed weekly until March 28, 1997. Additional data collection will include in-situ measurements of water pH and temperature, dissolved oxygen, and specific conductance. Totals of alkalinity, hardness and ammonia will also be measured in toxicity samples once samples are delivered to the laboratory.

V. SAMPLING METHODS 

Acute toxicity sampling will be conducted twice per week (Monday and Wednesday) at Orestimba Creek. Sampling for chronic toxicity will be conducted weekly on the San Joaquin River at Vernalis. One chronic sample constitutes the collection of a sample on days zero, two, and four of each week (e.g. Mon., Wed., and Fri.). Water collected on those days will be delivered the following day to the laboratory for testing and sample renewal. Chemical analyses will also be performed on each sample collected for both acute and chronic tests. Selected organophosphate and carbamate pesticides will be analyzed using three chemical "screens"

(Table 1)

Additionally, the Central Valley Regional Water Quality Board will conduct acute toxicity tests and chemical analyses on split samples from both Orestimba Creek and the San Joaquin River at Vernalis using different laboratories. A total of four samples, two from each site, will be examined for acute toxicity. The Regional Board will also analyze those same four samples for organophosphate pesticides. Scheduling for the collection of these additional samples will be concentrated near storm events.

A center channel water sample will be collected from bridges at each site. This will be done using a depth-integrated sampler (D-77) with a 3-liter Teflon® bottle and nozzle. Surface water sub-samples will be composited temporarily in a stainless steel container until the appropriate volume of water has been collected. This composited sample will then be stored on wet ice until delivered to the processing facility in West Sacramento. Immediately upon arrival at West Sacramento, the samples will be split into amber glass bottles using a Geotech® 10-port splitter then sealed with Teflon®-lined lids. Samples to be analyzed for organophosphate and carbamate pesticides will be acidified with 3N hydrochloric acid to a pH between 3.0 and 3.5. With the exception of diazinon, at this pH, most of the organophosphate and carbamate pesticides are sufficiently preserved. Therefore, diazinon will be analyzed from a separate, unacidified, split sample. Samples submitted for toxicity tests will not be acidified. Sufficient water will be collected during each sampling event to provide approximately three liters for chemical analysis, two liters each for both acute and chronic toxicity tests, and any additional water required for quality control samples. All samples will be stored at 4 C until delivered to the laboratories for toxicity testing and chemical analyses. In addition, samples submitted for toxicity testing will be delivered within 24 hours of collection.

Dissolved oxygen, pH, specific conductance, and water temperature will be measured in situ, at each site, during each sampling period. As part of the toxicity testing, the California Department of Fish and Game's Aquatic Toxicology Laboratory will measure and record other parameters of the delivered toxicity samples, including totals of alkalinity, hardness, and ammonia, and specific conductivity. These measurements will be made in the laboratory, thus they will not directly reflect on-site conditions.

VI. TOXICITY TESTING AND CHEMICAL ANALYSIS

Toxicity testing, conducted by the California Department of Fish and Game's Aquatic Toxicology Laboratory, will follow current U.S. Environmental Protection Agency procedures using the cladoceran Ceriodaphnia dubia (U.S. EPA, 1993). The Aquatic Toxicology Laboratory has been accredited by the California Department of Health Services' Environmental Laboratory Accreditation Program. Acute toxicity will be determined using a 96-hour, static-renewal bioassay in undiluted sample water. Chronic toxicity will be determined using a seven-day bioassay with C. dubia in undiluted sample water, and will follow current U.S. Environmental Protection Agency guidelines (U.S. EPA, 1994). For example, test organisms used in chronic testing will be subjected to sample water from day zero on the following day (day 1). Sample water collected on days two and four will then replace test water on days three and five, respectively. All bioassays will commence within 36 hours of sample collection. In addition, data will be reported to the project leader as percent survival on each day for the duration of the tests.

Chemical analysis will be performed by the California Department of Food and Agriculture Center for Analytical Chemistry. The reporting limit will be used to record the lowest concentration of analyte that the method can detect reliably in a matrix blank. The method titles and reporting limits for the study are listed in Table 1. Comprehensive chemical analytical methods will be provided in the final report.

VII. QUALITY ASSURANCE/QUALITY CONTROL

Toxicity 

To determine the variances within the primary laboratory, a quality assurance plan has been developed for this study (Barry, 1996). Briefly, additional samples from the acute toxicity monitoring site will be randomly submitted as blind matrix spikes and/or splits; they will be submitted as field samples for intra-laboratory comparison.

Chemical Analysis 

Quality control will be conducted in accordance with Standard Operating Procedure QAQC001.00. Ten percent of the total number of primary analyses will be submitted with field samples as blind matrix spikes and rinse blanks. The total number of samples is presented below.

Number of Samples for Toxicity Tests 

2 acute/week x 12 weeks...................................................................................................... 24

1 chronic sample (3 chronic sampling events)/week x 12 weeks.......................................... 12 

Total...................................................................................................................................... 36

Toxicity Testing Quality Control - see T. Barry (1996) 

Number of Chemical Analyses 

3 (organophosphate, carbamate, and diazinon) per acute sample:

3 analyses x 2 acute samples/week x 12 weeks.................................................................... 72

3 (organophosphate, carbamate, and diazinon) per chronic sampling event:

3 analyses x 3 chronic sampling events (1 chronic sample)/week x 12 weeks.................... 108 

Subtotal................................................................................................................................ 180

Quality Control

Continuing QC (approx. 10% of total chemical analyses)..................................................... 18 

Total..................................................................................................................................... 198

VIII. DATA ANALYSIS

Toxicity data will be used to establish baseline information on the occurrence of acute or chronic events at the Orestimba and Vernalis sites. A correlation matrix will be generated to investigate potential relationships between measured environmental parameters, discharge, toxic events, and chemical concentrations. Measured concentrations will be compared to various established water quality criteria including California Quantitative Response Limits, U.S. Environmental Protection Agency objectives, and California Department of Fish and Game suggested criteria. Pesticide concentrations will also be compared with acute and chronic LC50s for C. dubia to aid in interpreting toxicity test results. Toxicity data from the Regional Board's examinations will be used in the final analysis only if the selected laboratory's testing procedures follow current U.S. EPA guidelines.

With a fixed monitoring schedule, sample collection during storm events, when pesticide levels are typically highest, may not occur. Therefore, records of storm events will be kept, and an analysis of discharge, chemical data, and toxicity will be discussed in relation to sampling periods. Depending on preliminary analysis results, further analysis may include logistic regression. Quality control results from toxicity tests will be used to develop a quality assurance plan for future aquatic toxicity studies.

IX. TIME TABLE

Field Sampling - December, 1996 and application onset through

March 28, 1997

Toxicity Testing and Chemical Analysis - November 1996 through April 1997

Preliminary Memorandum - May 1997

Final Report - August 1997

X. REFERENCES 

Ando, C. 1996. Investigation of possible management practices to reduce dormant spray runoff from soil plots (study protocol). California EPA/Department of Pesticide Regulation, Environmental Hazards Assessment Program. May 24, 1996.

Anonymous, 1996. Everett Souza shares HUA story. Westside Water, March 1996.

Barry, T.A. 1996. Protocol for obtaining a preliminary characterization of intralaboratory precision of acute toxicity tests performed on ambient water samples. California EPA/Department of Pesticide Regulation, Environmental Hazards Assessment Program. December, 1996.

Biermann, H. 1996. Study 152: Effect of ground cover on dormant spray runoff from orchards (study protocol). California EPA/Department of Pesticide Regulation, Environmental Hazards Assessment Program. November 1996.

CVRWQCB, 1994. (Central Valley Regional Water Quality Control Board) Water quality control plan (Basin Plan): central valley region, Sacramento River and San Joaquin River Basins. December 9, 1994.

Domagalski, J.L. 1995. Nonpoint sources of pesticides in the San Joaquin River, California:input from winter storms, 1992-93. U.S. Geological Survey Open-File Report 95-165. National Water-Quality Assessment Program. Sacramento, California.

Foe, C. 1995. Insecticide concentrations and invertebrate bioassay mortality in agricultural return water from the San Joaquin Basin. Central Valley Regional Water Quality Control Board. December 1995.

Foe, C. and V. Connor. 1991. San Joaquin Watershed bioassay results, 1988-90. Central Valley Regional Water Quality Control Board. July 1991.

Foe, C. and R. Sheipline. 1993. Pesticides in surface water from applications on orchards and alfalfa during the winter and spring of 1991-92. Central Valley Regional Water Quality Control Board. February 1993.

Kuivila, K. And C. Foe. 1995. Concentrations, transport and biological effects of dormant spray pesticides in the San Francisco estuary, California. Environ. Toxicol. Chem. 14(7):1141-1150.

Edwards, T.K. and D.G. Glysson. 1988. Field methods for measurement of fluvial sediment: U.S. Geological Survey Open-File Report 86-531. p. 61-64.

Ross, L.J. 1992. Preliminary results of the San Joaquin River study; Winter 1991-92.

Memorandum to Kean Goh, Environmental Hazards Assessment Program, California EPA, Department of Pesticide Regulation. May 22, 1992.


Ross, L.J. 1993. Preliminary results of the San Joaquin River study; Winter 1992-93.

Memorandum to Kean Goh, Environmental Hazards Assessment Program, California EPA, Department of Pesticide Regulation. September 23, 1993.

Ross, L.J. 1995. Reducing dormant spray runoff from orchards (study protocol). Department of Pesticide Regulation/California EPA, Environmental Hazards Assessment Program. November 15, 1995.

Standard Operating Procedure Number: QAQC001.00. 1996. Chemistry Laboratory Quality Control. California EPA, Department of Pesticide Regulation, Environmental Hazards Assessment Program.

U.S. Environmental Protection Agency. 1993. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. 4th ed. EPA/600/4-90/027F. August 1993.

U.S. Environmental Protection Agency. 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. 3rd ed.

EPA-600-4-91-002. July 1994.


Zalom, F.G., R.A. Van Steenwyk, W.J. Bentley, R. Coviello, R.E. Rice, W.W. Barnett, C. Pickel, M.M. Barnes, B.L. Teviotdale, W.D. Gubler, and M.V. McKenry. 1995. Almond pest management guidelines. Univ. Of California, Division of Agriculture and Natural Resources, UCPMG Publication 1.




















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Table 1. California Department of Food and Agriculture, Laboratory Services Branch: organophosphate and carbamate pesticide screens for the San Joaquin River toxicity monitoring study.
Organophosphate Pesticides in Surface Water 

by GC 

Method: GC/FPD
N-Methyl Carbamate in Surface Water by HPLC 

Method: HPLC/Post Column-fluorescence


Compound
Reporting Limit 

(mg/L)


Compound
Reporting Limit 

(mg/L)
Chlorpyrifos 0.04 Carbaryl 0.05
Diazinon1 0.04 Carbofuran 0.05
Dimethoate (Cygon) 0.05
Fonofos 0.05
Malathion 0.05
Methidathion 0.05
Methyl parathion 0.05
Phosmet 0.05
1) Diazinon will be analyzed from a separate, unpreserved, split sample. Other chemical samples will be preserved with 3N HCl to a pH of 3-3.5 to retard analyte degradation. See text.