Brine Shrimp Bioassay Procedure For Determining Produced Water Toxicity
Authors: Palmer L.L. 
Published: December 2007

Abstract
Oil field produced waters must comply with environmental protection regulations when discharged to the environment. One of the parameters produced voters may be required to meet is toxicity as determined by laboratory bioassays. A bioassay procedure using brine shrimp (Artemia salina) has procedure using brine shrimp (Artemia salina) has been developed that cam be used on produced waters throughout the country and independent of the time of year.

Introduction
Estimating the toxicity of an effluent to aquatic life requires an analytical procedure known as a bioassay. Of the several types conducted, the static laboratory bioassay is the most common.

Static Laboratory Bioassays
The static laboratory bioassay consists basically of placing a known number of test organisms in incremental dilutions of the effluent in question. After a specified time period, the surviving organisms are counted and a median lethal concentration (LC50) is calculated. An LC50 is defined as the percent by volume that kills 50% of the test organisms. Thus, a 96-hr LC50 of 67% means that a mixture of 67% effluent and 33% clean water killed 50% of the test organisms in 96 hours. The LC50 of an effluent increases as its toxicity decreases.

Bioassay Organisms
Fish such as sticklebacks (Gasterosteus aculeatus) and killifish (Fundulus parvipinnis) are traditional bioassay organisms, but can generally survive satisfactorily only in waters of less than 4% salinity. Table 1 shows that the salinities of produced waters vary from about 1.5% (15,000 mg/liter produced waters vary from about 1.5% (15,000 mg/liter total dissolved solids) to over 13.5% (135,000 mg/ liter) in some Gulf Coast fields. Thus, an otherwise nontoxic Gulf Coast produced water could appear extremely toxic to fish. The brine shrimp (Artemia saline), however, can flourish at salinities between 0.2% (2,000 mg/liter) and 20% (200,000 mg/liter).
This paper presents a procedure for determining a 48-hr LC50 for produced water using brine shrimp.

BRINE SHRIMP
Dried eggs of the brine shrimp Artemia salina are readily available throughout the country independent of seasonal conditions. The Artemia are hatched in the laboratory under controlled conditions, ensuring uniformity of organisms. Newly-hatched Artemia (nauplii) are about 2 mm long, an ideal bioassay size: large enough to see without high magnification, yet small enough not to require the large holding tanks and extensive work space needed with fish.
Artemia nauplii have branch-like appendages that also serve as gills (Figure 1) and continually flutter when the organism is healthy. No movement indicates death, making live versus dead determinations during the bioassay relatively simple.
As a result of their adaptability to a wide range of salinities, general availability, small size, and minimal equipment needs, Artemia nauplii were chosen as the organism with which to develop a produced water bioassay procedure. Brine shrimp produced water bioassay procedure. Brine shrimp bioassay procedures previously published have been for determining the toxicity of oil dispersants and for evaluating application; under the EPA's ocean Disposal Permit Program.

EQUIPMENT and FACILITY REQUIREMENTS

Laboratory
Any laboratory is acceptable that can be temperature-controlled to 20 degrees C plus or minus 2 degrees C for the 48 hr test period. This requirement is critical to satisfactory and reproducible bioassay results.